Nanobodies against tumor necrosis factor-alpha

ABSTRACT

The present invention relates to improved Nanobodies™ against Tumor Necrosis Factor-alpha (TNF-alpha), as well as to polypeptides comprising or essentially consisting of one or more of such Nanobodies. The invention also relates to nucleic acids encoding such Nanobodies and polypeptides; to methods for preparing such Nanobodies and polypeptides; to host cells expressing or capable of expressing such Nanobodies or polypeptides; to compositions comprising such Nanobodies, polypeptides, nucleic acids or host cells; and to uses of such Nanobodies, such polypeptides, such nucleic acids, such host cells or such compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/644,890, filed Jul. 10, 2017, which is a continuation of U.S.application Ser. No. 14/709,870, which is a continuation of U.S.application Ser. No. 14/191,907, filed Feb. 27, 2014 and now issued asU.S. Pat. No. 9,067,991, which is a divisional of U.S. application Ser.No. 11/920,677, filed Jul. 30, 2010 and now issued as U.S. Pat. No.8,703,131, which is a national stage filing under 35 U.S.C. § 371 ofinternational application PCT/EP2006/004678, filed May 17, 2006, andclaims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication Ser. No. 60/682,332, filed May 18, 2005, the disclosures ofeach of which are incorporated by reference herein in their entirities.

INCORPORATION BY REFERENCE

This application contains a Sequence Listing which has been filedelectronically in ASCII and is hereby incorporated by reference in itsentirety. This ASCII copy, created on Jun. 30, 2021, is namedA084870008US06-SUBSEQ-CRP and is 225,035 bytes in size.

The present invention relates to improved Nanobodies™ against TumorNecrosis Factor-alpha (TNF-alpha), as well as to polypeptides comprisingor essentially consisting of one or more of such Nanobodies. [Note:Nanobody™, Nanobodies™ and Nanoclone™ are trademarks of Ablynx N.V.]

The invention also relates to nucleic acids encoding such Nanobodies andpolypeptides; to methods for preparing such Nanobodies and polypeptides;to host cells expressing or capable of expressing such Nanobodies orpolypeptides; to compositions comprising such Nanobodies, polypeptides,nucleic acids or host cells; and to uses of such Nanobodies, suchpolypeptides, such nucleic acids, such host cells or such compositions,in particular for prophylactic, therapeutic or diagnostic purposes, suchas the prophylactic, therapeutic or diagnostic purposes mentioned below.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description hereinbelow.

WO 04/041862 by applicant relates to Nanobodies against TNF-alpha and tothe preparation and use thereof, in particular for the prevention and/ortreatment of diseases and disorders associated with and/or mediated byTNF-alpha, such as inflammation, rheumatoid arthritis, Crohn's disease,ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis,addison's disease, autoimmune hepatitis, autoimmune parotitis, diabetestype 1, epididymitis, glomerulonephritis, Graves' disease,Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemiclupus erythematosus, male infertility, multiple sclerosis, myastheniagravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies,thyroiditis, and vasculitis.

The anti-TNF Nanobodies according to WO 04/041862 may be humanized andmay be monovalent or multivalent, the latter of which leads to increasedaffinity for TNF. The anti-TNF Nanobodies™ according to WO 04/041862 mayalso be multispecific, and may in particular be in the form of amultispecific construct comprising two or more Nanobodies against TNFand a further Nanobody directed against a serum protein such as humanserum albumin, which leads to an increased half-life in vivo.

WO 04/041862 also relates to methods for the preparation of the anti-TNFNanobodies, to nucleic acids or constructs encoding the anti-TNFNanobodies, as well as to pharmaceutical compositions comprising theanti-TNF Nanobodies, which may be suitable for intravenous,subcutaneous, oral, sublingual, topical, nasal, vaginal or rectaladministration, or for administration by inhalation. The anti-TNFNanobodies according to WO 04/041862 may also be used for diagnosticpurposes, optionally in the form of a kit-of-parts.

EP 0 486 526 describes TNF-alpha binding ligands against a specificepitope of TNF. Among the binding ligands, single domain antibodies(“dAbs”) are mentioned.

Reiter et al., J. Mol. Biol. (1999), 290, 685-698 describe single domainantibodies against TNF-alpha obtained from a randomized phage displaylibrary that was generated starting from a VH domain scaffold from amouse hybridoma.

WO 00/29004 describes murine single domain antibodies (“microbodies”)against TNF-alpha.

WO 04/003019 inter alia describes ligands comprising a first bindingdomain against TNF-alpha and a second binding domain against a serumprotein such as serum albumin.

It is a general object of the present invention to provide Nanobodiesagainst TNF-alpha, in particular against human TNF-alpha.

In particular, it is an object of the present invention to provideNanobodies against TNF-alpha, in particular against human TNF-alpha, andto provide proteins or polypeptides comprising the same, that aresuitable for therapeutic and/or diagnostic use, and in particular forthe prevention, treatment and/or diagnosis of one or more diseases anddisorders associated with and/or mediated by TNF-alpha such as thosementioned above, and/or that can be used in the preparation of apharmaceutical composition for the prevention and/or treatment of one ormore diseases associated with and/or mediated by TNF-alpha, such asthose mentioned above.

More in particular, it is an object of the invention to provideNanobodies against TNF-alpha, and to provide proteins and polypeptidescomprising the same, that are either an alternative to the Nanobodiesand polypeptides against TNF-alpha described in WO 04/041862 and/or thathave one or more improved properties or characteristics, compared to theNanobodies and polypeptides against TNF-alpha described in WO 04/041862.

More in particular, it is an object of the invention to provideNanobodies against TNF-alpha, and to provide proteins or polypeptidescomprising the same, that are improved compared to the Nanobodies andpolypeptides against TNF-alpha described in WO 04/041862 with respect toone or more of the following properties or characteristics:

-   -   increased affinity for TNF-alpha, either in a monovalent format,        in a multivalent format (for example in a bivalent format)        and/or in a multispecific format (for example one of the        multispecific formats described in WO 04/041862 or hereinbelow);    -   better suitability for formatting in a multivalent format (for        example in a bivalent format);    -   better suitability for formatting in a multispecific format (for        example one of the multispecific formats described in WO        04/041862 or hereinbelow);    -   improved suitability or susceptibility for “humanizing”        substitutions (as defined herein); and/or    -   less immunogenicity, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described in WO 04/041862 or hereinbelow) in a        monovalent format;    -   increased stability, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described in WO 04/041862 or hereinbelow) in a        monovalent format;    -   increased specificity towards TNF-alpha, either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described in WO 04/041862 or hereinbelow)        in a monovalent format;    -   decreased or where desired increased cross-reactivity with        TNF-alpha from different species;        and/or    -   one or more other improved properties desirable for        pharmaceutical use (including prophylactic use and/or        therapeutic use) and/or for diagnostic use (including but not        limited to use for imaging purposes), either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described in WO 04/041862 or hereinbelow).

These objects are achieved by the Nanobodies, proteins and polypeptidesdescribed herein. These Nanobodies are also referred to herein as“Nanobodies of the invention”; and these proteins and polypeptides arealso collectively referred to herein “polypeptides of the invention”.

Since the Nanobodies and polypeptides described herein are mainlyintended for therapeutic and/or diagnostic use, they are directedagainst (as defined herein) human TNF-alpha. It is however not excluded(but also not required) that Nanobodies and polypeptides describedherein show cross-reactivity with TNF-alpha from one or more otherspecies of warm-blooded animals, for example with TNF-alpha from one ormore other species of primates and/or with TNF-alpha from one or morespecies of animals that are often used in animal models for diseases(for example mouse, rat, rabbit, pig or dog), and in particular inanimal models for diseases and disorders associated with TNF-alpha (suchas the species and animal models mentioned herein). In this respect, itwill be clear to the skilled person that such cross-reactivity, whenpresent, may have advantages from a drug development point of view,since it allows the Nanobodies and polypeptides against human TNF-alphato be tested in such disease models.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of TNF-alphaagainst which the Nanobodies and polypeptides of the invention aredirected.

However, in a preferred embodiment, the Nanobodies, proteins orpolypeptides described herein are directed against and/or can bind to anepitope of TNF-alpha that lies in and/or forms part of the TNF receptorbinding site(s) (e.g. the binding sites for the TNF-RI, THF-RII, alsoknown as p55 or p75). As is well known in the art, a TNF trimercomprises three receptor binding sites, which are essentially equivalentand which are formed by/at the interface of two TNF monomers within theTNF trimer. For example, the Nanobodies, proteins or polypeptidesdescribed herein are preferably directed against and/or can bind to anepitope of TNF-alpha that comprises the following amino acid residues ofTNF-alpha: Gln at position 88, Lys at position 90, and/or Glu atposition 146).

In particular, the Nanobodies, proteins or polypeptides described hereinare directed against and/or can bind to an epitope of the TNF-alphatrimer, which lies in and/or forms part of the TNF receptor bindingsite(s). For example, the Nanobodies, proteins or polypeptides describedherein may be directed against and/or can bind to an epitope of theTNF-alpha trimer that comprises the following amino acid residues: Glnat position 88 and Lys at position 90 on a first TNF monomer (referredto herein as “monomer A”), and Glu at position 146 on a second TNFmonomer (referred to herein as “monomer B”) (in which Monomer A andMonomer B together, in the TNF trimer, form the TNF receptor bindingsite(s)).

More particularly, the Nanobodies, proteins or polypeptides describedherein may be directed against and/or can bind to an epitope of theTNF-alpha trimer that comprises the aforementioned amino acids (Gln atposition 88 in monomer A; Lys at position 90 in monomer A and Glu atposition 146 in monomer B), and in addition at least one, preferably twoor more, more preferably 5 or more, and preferably all or essentiallyall, of the following amino acid residues of TNF-alpha monomer A: Gly atposition 24, Gln at position 25, Thr at position 72, His at position 73,Val at position 74, Leu at position 75, Thr at position 77, Thr atposition 79, Be at position 83, Thr at position 89, Val at position 91.Asn at position 92, Ile at position 97, Arg at position 131, Glu atposition 135, Ile at position 136, Asn at position 137, Arg at position138, Pro at position 139, Asp at position 140 and the following residuesin monomer B: Pro at position 20, Arg at position 32, Lys at position65, Lys at position 112, Tyr at position 115, Ala at position 145, Serat position 147.

Alternatively, the Nanobodies, proteins or polypeptides described hereinmay be directed against and/or can bind to an epitope of TNF-alpha thatcomprises the aforementioned amino acids (Gln at position 88 in monomerA; Lys at position 90 in monomer A and Glu at position 146 in monomerB), and in addition at least one, preferably two or more, morepreferably 5 or more and preferably all or essentially all, of thefollowing amino acid residues of TNF-alpha monomer A: Leu at position75, Thr at position 77, Thr at position 79, Be at position 80, Ser atposition 81, Tyr at position 87, Thr at position 89, Val at position 91,Asn at position 92, Ser at position 95, Be at position 97, Glu atposition 135, Ile at position 136, Asn at position 137 and the followingresidues in monomer B: Ala at position 33, Ala at position 145, Ser atposition 147.

Such epitope can be delineated from structural analysis of the nanobodycrystallized in complex with the TNF molecule, or from other approachessuch as epitope mapping via pepscan analysis.

By comparison, from crystallographic data (not shown), it can be seenthat the Nanobody 3E from WO 04/041862 binds to a different epitope(i.e. an epitope comprising Tyr at position 141, Asp at position 140,Gln at position 67, Gly at position 24 and Glu at position 23) than thepreferred epitope of the invention.

Thus, in another aspect, the present invention relates to animmunoglobulin variable domain (or a suitable fragment thereof) that canbind to an epitope of TNF-alpha that lies in and/or forms part of theTNF receptor binding site, and preferably to an epitope that comprisesat least one, preferably two or more, and preferably all, of thefollowing amino acid residues of TNF-alpha: Gln at position 88; Lys atposition 90 and Glu at position 146. Such an immunoglobulin variabledomain is preferably a heavy chain variable domain or a light chainvariable domain, and in particular a heavy chain variable domain, whichmay be any mammalian heavy chain variable domain, including but notlimited to human heavy chain variable domains, mouse heavy chainvariable domains and Camelid heavy chain variable domains (such as theheavy chain variable domains from Camelid 4-chain immunoglobulins or theheavy chain variable domains (VHH domains) from so-called heavy chainantibodies). The immunoglobulin variable domain is preferably a domainantibody or single domain antibody or suitable for use as a (single)domain antibody. Most preferably, the immunoglobulin variable domain isa Nanobody (as defined herein), and some preferred, but non-limitingexamples of Nanobodies that are suitable for use in this aspect of theinvention are PMP1C2 (TNF1, SEQ ID NO:52) and PMP5F10 (TNF3, SEQ ID NO:60), as well as humanized and other variants thereof (as furtherdescribed herein).

The aforementioned immunoglobulin variable domain may also be humanized(as for example, and without limitation) described herein with respectto Nanobodies. The invention also relates to proteins and polypeptidesthat comprise or essentially consist of such immunoglobulin variabledomains, which may for example be as defined herein. Alternatively, suchvariable domains may form part of ScFv constructs, dual-specificconstructs, chimeric antibody or antibody structures and otherimmunoglobulin constructs, as for example reviewed by Hoogenboom (NatureBiotechnology (1997), 15:125-126). Preferably, however, theimmunoglobulin variable domains directed against the above epitope areNanobodies, in which case the proteins and polypeptides comprising suchNanobodies may be as further described herein.

Thus, some preferred aspects of the invention relate to:

-   I) A Nanobody which is directed against the same epitope on the    trimer of TNF-alpha as Nanobody TNF1 (SEQ ID NO: 52).-   II) A Nanobody which is directed against the same epitope on the    trimer of TNF-alpha as Nanobody TNF3 (SEQ ID NO: 60).-   III) A Nanobody which is directed against an epitope of the trimer    of TNF-alpha that at least comprises the following amino acid    residues: Gln at position 88 in monomer A; Lys at position 90 in    monomer A and Glu at position 146 in monomer B.-   IV) A Nanobody which is directed against an epitope of the trimer of    TNF-alpha that comprises the following amino acid residues: Gln at    position 88 in monomer A; Lys at position 90 in monomer A and Glu at    position 146 in monomer B; and that further comprises at least    comprises at least one, preferably two or more, more preferably 5 or    more, and preferably all or essentially all, of the following amino    acid residues of TNF-alpha monomer A: Gly at position 24, Gln at    position 25, Thr at position 72, His at position 73, Val at position    74, Leu at position 75, Thr at position 77, Thr at position 79, Ile    at position 83, Thr at position 89, Val at position 91. Asn at    position 92, Be at position 97, Arg at position 131, Glu at position    135, Ile at position 136, Asn at position 137, Arg at position 138,    Pro at position 139, Asp at position 140 and the following residues    in monomer B: Pro at position 20, Arg at position 32, Lys at    position 65, Lys at position 112, Tyr at position 115, Ala at    position 145, Ser at position 147.-   V) A Nanobody which is directed against an epitope of the trimer of    TNF-alpha that comprises the following amino acid residues: Gln at    position 88 in monomer A; Lys at position 90 in monomer A and Glu at    position 146 in monomer B; and that further comprises at least one,    preferably two or more, more preferably 5 or more, and preferably    all or essentially all, of the following amino acid residues of    TNF-alpha monomer A Leu at position 75, Thr at position 77, Thr at    position 79, Ile at position 80, Ser at position 81, Tyr at position    87, Thr at position 89, Val at position 91, Asn at position 92, Ser    at position 95, Be at position 97, Glu at position 135, Ile at    position 136, Asn at position 137 and the following residues in    monomer B: Ala at position 33, Ala at position 145, Ser at position    147.    -   A Nanobody in accordance with any of I) to V) above, which has a        K_(off) rate for TNF of better than 2.10⁻³ (1/s), preferably        better than 1.10⁻³.    -   A Nanobody in accordance with any one of I) to V) above, which        has an EC50 value in the cell-based assay using KYM cells        described in Example 1, under 3), of WO 04/041862 that is better        than the EC50 value of Nanobody VHH 3E (SEQ ID NO:4) of WO        04/041862 in the same assay; or a humanized variant of such a        Nanobody.    -   A Nanobody in accordance with any one of I) to V) above, which        has an EC50 value in the cell-based assay using KYM cells        described in Example 1, under 3), of WO 04/041862 that is better        than 12 nM; or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with any one of I) to V) above, which        has an EC50 value in the cell-based assay using KYM cells        described in Example 1, under 3), of WO 04/041862 that is better        than 5 nM; or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with any one of I) to V) above, which        has an EC50 value in the cell-based assay using KYM cells        described in Example 1, under 3), of WO 04/041862 that is better        than 3 nM; or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with any one of I) to V) above, which        is a humanized Nanobody.        and some preferred aspects of this embodiment relate to:    -   A Nanobody in accordance with any one of I) to V) above, which        is a GLEW-class Nanobody.    -   A Nanobody in accordance with any one of I) to V) above, which        is a humanized GLEW-class Nanobody.    -   A Nanobody in accordance with any one of I) to V) above, which        contains an arginine residue (R) at position 103.    -   A Nanobody in accordance with any one of I) to V) above, which        contains an arginine residue (R) at position 103, and which is        humanized    -   A Nanobody in accordance with any one of I) to V) above, which        is a GLEW-class Nanobody, and which contains an arginine        residue (R) at position 103, and which is humanized.    -   A Nanobody in accordance with any one of I) to V) above, which        contains a leucine residue (L) at position 108.    -   A Nanobody in accordance with any one of I) to V) above, which        has at least 80%, preferably at least 90%, more preferably at        least 95%, even more preferably at least 99% sequence identity        (as defined herein) with one of the amino acid sequences of SEQ        ID NO's 52 (TNF1), 76 (TNF13), 77 (TNF14), 95 (TNF29) or 96        (TNF30)    -   A Nanobody in accordance with any one of I) to V) above, in        which    -   a) CDR1 comprises:        -   the amino acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence DYWMY (SEQ ID NO:            164);    -   and    -   b) CDR2 comprises:        -   the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 232);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 232); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 232);    -   and    -   c) CDR3 comprises:        -   the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence SPSGFN (SEQ ID            NO: 300).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:        164).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG        (SEQ ID NO: 232).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO:        300).    -   A Nanobody in accordance with any one of I) to V) above, in        which:        -   CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:            164); and CDR3 comprises the amino acid sequence SPSGFN (SEQ            ID NO: 300); or        -   CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:            164); and CDR2 comprises the amino acid sequence            EINTNGLITKYPDSVKG (SEQ ID NO: 232); or        -   CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 232); and CDR3 comprises the amino acid sequence            SPSGFN (SEQ ID NO: 300)    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:        164); and CDR3 comprises the amino acid sequence SPSGFN (SEQ ID        NO: 300).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:        164); CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG        (SEQ ID NO: 232) and CDR3 comprises the amino acid sequence        SPSGFN (SEQ ID NO: 300).    -   A Nanobody in accordance with any one of I) to V) above, in        which    -   a) CDR1 is:        -   the amino acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence DYWMY (SEQ ID NO:            164);    -   and in which:    -   b) CDR2 is:        -   the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 232);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 232); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 232);    -   and in which    -   c) CDR3 is:        -   the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SPSGFN(SEQ ID NO: 300); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SPSGFN (SEQ ID NO:            300).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID        NO: 232).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR3 is the amino acid sequence SPSGFN (SEQ OD NO: 300)    -   A Nanobody in accordance with any one of I) to V) above, in        which:        -   CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); and            CDR3 is the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); and            CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID            NO:232); or        -   CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID            NO: 232); and CDR3 is the amino acid sequence SPSGFN (SEQ ID            NO: 300)    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164);        and CDR3 is the amino acid sequence SPSGFN (SEQ ID NO: 300).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164);        CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID        NO: 232) and CDR3 is the amino acid sequence SPSGFN (SEQ ID NO:        300).    -   A Nanobody in accordance with any one of I) to V) above, in        which        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).            and some other preferred aspects of this embodiment relate            to:    -   A Nanobody in accordance with any one of I) to V) above, which        is a KERE-class Nanobody    -   A Nanobody in accordance with any one of I) to V) above, which        is a humanized KERE-class Nanobody    -   A Nanobody in accordance with any one of I) to V) above, which        has at least 80%, preferably at least 90%, more preferably at        least 95%, even more preferably at least 99% sequence identity        (as defined herein) with one of the amino acid sequences of SEQ        ID NO's 50 (TNF3), 83 (TNF20), 85 (TNF21), 85 (TNF22), 96        (TNF23) or 98 (TNF33).    -   A Nanobody in accordance with any one of I) to V) above, in        which    -   a) CDR1 is:        -   the amino acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence NYYMG (SEQ ID NO:            172);    -   and    -   b) CDR2 is:        -   the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or an            amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence            NISWRGYNIYYKDSVKG;    -   and    -   c) CDR3 is:        -   the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SILPLSDDPGWNTY (SEQ            ID NO: 308).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID        NO: 240).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR3 is the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO:        308).    -   A Nanobody in accordance with any one of I) to V) above, in        which:        -   CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); and            CDR3 is the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO:            308); or        -   CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); and            CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID            NO: 240); or        -   CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID            NO: 240); and CDR3 is the amino acid sequence SILPLSDDPGWNTY            (SEQ ID NO: 308).    -   A Nanobody in accordance with any one of I) to V) above, in        which CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172);        CDR2 is the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308)        and CDR3 is the amino acid sequence ILPLSDDPGWNTY (SEQ ID NO:        436).    -   A Nanobody in accordance with any one of I) to V) above, in        which        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).            and with yet some other particularly preferred aspects            being:-   VI) A protein or polypeptide, which comprises a or essentially    consists a Nanobody in accordance with any one of I) to V) above.-   VII) A protein or polypeptide, which comprises or essentially    consists of at least one Nanobody in accordance with any one of I)    to V) above.-   VIII) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of I) to V) above.-   IX) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of I) to V) above, and which is such that    said protein or polypeptide, upon binding to a TNF trimer, is    capable inhibiting or reducing the TNF receptor crosslinking that is    mediated by said TNF trimer and/or the signal transduction that is    mediated by such receptor crosslinking.-   X) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of I) to V) above, and which is capable of    intramolecular binding to at least two TNF receptor binding sites on    a TNF trimer.-   XI) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of I) to V) above, linked via a suitable    linker.-   XII) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of I) to V) above, linked via a suitable    linker, and which is pegylated.-   XIII) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of I) to V) above, and which further    comprises at least one Nanobody directed against human serum    albumin.-   XIV) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of I) to V) above, and which further    comprises at least one Nanobody directed against human serum    albumin, and which is such that said protein or polypeptide, upon    binding to a TNF trimer, is capable inhibiting or reducing the TNF    receptor crosslinking that is mediated by said TNF trimer and/or the    signal transduction that is mediated by such receptor crosslinking.-   XV) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of I) to V) above, and which further    comprises at least one Nanobody directed against human serum albumin    and which is capable of intramolecular binding to at least two TNF    receptor binding sites on a TNF trimer.-   XVI) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of I) to V) above, and which further    comprises one Nanobody directed against human serum albumin, in    which each of the two Nanobodies in accordance with any one of I)    to V) above is linked, optionally via a suitable linker, to the one    Nanobody directed against human serum albumin.-   XVII) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of I) to V) above, and which further    comprises one Nanobody directed against human serum albumin, in    which each of the two Nanobodies in accordance with any one of I)    to V) above is linked, optionally via a suitable linker, to the one    Nanobody directed against human serum albumin, and which is such    that said protein or polypeptide, upon binding to a TNF trimer, is    capable inhibiting or reducing the TNF receptor crosslinking that is    mediated by said TNF trimer and/or the signal transduction that is    mediated by such receptor crosslinking.-   XVIII) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of I) to V) above, and which further    comprises one Nanobody directed against human serum albumin, in    which each of the two Nanobodies in accordance with any one of I)    to V) above is linked, optionally via a suitable linker, to the one    Nanobody directed against human serum albumin, and which is capable    of intramolecular binding to at least two TNF receptor binding sites    on a TNF trimer.    -   A protein or polypeptide in accordance with any one of VI)        to XVIII) above, in which the at least one Nanobody directed        against human serum albumin is a humanized Nanobody.    -   A protein or polypeptide in accordance with any one of VI)        to XVIII) above, in which the at least one Nanobody directed        against human serum albumin is a humanized variant of the        Nanobody ALB 1 (SEQ ID NO: 63).    -   A protein or polypeptide in accordance with any one of VI)        to XVIII) above, in which the at least one Nanobody directed        against human serum albumin is a chosen from the group        consisting of ALB 3 (SEQ ID NO: 87), ALB 4 (SEQ ID NO: 88), ALB        5 (SEQ ID NO: 89), ALB 6 (SEQ ID NO: 100), ALB 7 (SEQ ID NO:        101), ALB 8 (SEQ ID NO: 102) ALB 9 (SEQ ID NO: 103) and ALB 10        (SEQ ID NO: 104).    -   A protein or polypeptide in accordance with any one of VI)        to XVIII) above, in which the at least one Nanobody directed        against human serum albumin is ALB 8.    -   A protein or polypeptide in accordance with any one of VI)        to XVIII) above, which comprises or essentially consists of two        humanized Nanobodies in accordance with any one of I) to V)        above, and one humanized variant of the Nanobody ALB 1 (SEQ ID        NO: 63).

It should be noted that when a Nanobody is mentioned above as being “inaccordance with any one of I) to V) above”, it is at least according toone of I) to V), may be according to two or more of I) to V), and mayalso include any one or more of the other aspects that are indicated asbeing “in accordance with any one of I) to V) above. Similarly, when aprotein or polypeptide is mentioned above as being “in accordance withany one of VI) to XVIII) above”, it is at least according to one of VI)to XVIII), may be according to two or more of VI) to XVIII), and mayalso include any one or more of the other aspects that are indicated asbeing “in accordance with any one of VI) to XVIII) above.

It is also within the scope of the invention that, where applicable, aNanobody or polypeptide of the invention can bind to two or moreantigenic determinants, epitopes, parts, domains, subunits orconfirmations of TNF-alpha. In such a case, the antigenic determinants,epitopes, parts, domains or subunits of TNF-alpha to which theNanobodies and/or polypeptides of the invention bind may be theessentially same (for example, if TNF-alpha contains repeated structuralmotifs or is present as a multimer) or may be different (and in thelatter case, the Nanobodies and polypeptides of the invention may bindto such different antigenic determinants, epitopes, parts, domains,subunits of TNF-alpha with an affinity and/or specificity which may bethe same or different). Also, for example, when TNF-alpha exists in anactivated conformation and in an inactive conformation, the Nanobodiesand polypeptides of the invention may bind to either one of theseconformation, or may bind to both these conformations (i.e. with anaffinity and/or specificity which may be the same or different). Also,for example, the Nanobodies and polypeptides of the invention may bindto a conformation of TNF-alpha in which it is bound to a pertinentligand, may bind to a conformation of TNF-alpha in which it not bound toa pertinent ligand, or may bind to both such conformations (again withan affinity and/or specificity which may be the same or different).

It is also expected that the Nanobodies and polypeptides of theinvention will generally bind to all naturally occurring or syntheticanalogs, variants, mutants, alleles, parts and fragments of TNF-alpha,or at least to those analogs, variants, mutants, alleles, parts andfragments of TNF-alpha that contain one or more antigenic determinantsor epitopes that are essentially the same as the antigenicdeterminant(s) or epitope(s) to which the Nanobodies and polypeptides ofthe invention bind in TNF-alpha (e.g. in wild-type TNF-alpha). Again, insuch a case, the Nanobodies and polypeptides of the invention may bindto such analogs, variants, mutants, alleles, parts and fragments with anaffinity and/or specificity that are the same as, or that different from(i.e. higher than or lower than), the affinity and specificity withwhich the Nanobodies of the invention bind to (wild-type) TNF-alpha. Itis also included within the scope of the invention that the Nanobodiesand polypeptides of the invention bind to some analogs, variants,mutants, alleles, parts and fragments of TNF-alpha, but not to others.

Generally, the Nanobodies and polypeptides of the invention will atleast bind to those forms (including monomeric, multimeric andassociated forms) that are the most relevant from a biological and/ortherapeutic point of view, as will be clear to the skilled person.

Also, as TNF-alpha exists in a monomeric form and in multimeric forms,and in particular in trimeric form, it is within the scope of theinvention that the Nanobodies and polypeptides of the invention onlybind to TNF-alpha in monomeric form, or that the Nanobodies andpolypeptides of the invention in addition also bind to one or more ofsuch multimeric forms, such as the trimeric form of TNF; or may onlybind to such a multimeric (e.g. trimeric) form. Thus, generally, when inthis description reference is made to a Nanobody, protein or polypeptidethat is directed to TNF-alpha, it should be understood that this alsocomprises Nanobodies directed against TNF-alpha in its trimeric form(including but not limited to Nanobodies against the receptor bindingsites (e.g. the binding sites for the TNF-RI, THF-RII, also known as p55or p′75) of such a trimer). In all these cases, the Nanobodies andpolypeptides of the invention may bind to such multimers or associatedprotein complexes with an affinity and/or specificity that may be thesame as or different from (i.e. higher than or lower than) the affinityand/or specificity with which the Nanobodies and polypeptides of theinvention bind to TNF-alpha in its monomeric and non-associated state.

Also, generally, polypeptides of the invention that contain two or moreNanobodies directed against TNF-alpha may bind with higher avidity thanthe corresponding monomeric Nanobody or Nanobodies.

For example, and without limitation, a multivalent (as defined herein)protein or polypeptide that contains two or more Nanobodies that aredirected against different epitopes of TNF-alpha multivalent (as definedherein) protein or polypeptide that contains two or more Nanobodies thatare directed against different epitopes of TNF-alpha may bind toTNF-alpha with higher avidity than the corresponding monomers.

More importantly, a multivalent (as defined herein) protein orpolypeptide that contains two or more Nanobodies that are directedagainst TNF-alpha may (and usually will) bind with higher avidity to amultimer of TNF-alpha than to a monomer of TNF-alpha, and will usuallyalso bind with higher avidity than the corresponding monomericNanobodies. In such a multivalent protein or polypeptide, the two ormore Nanobodies may for example be directed against the same epitopes,substantially equivalent epitopes, or different epitopes. In oneembodiment of such a multivalent protein or polypeptide, the two or moreNanobodies may be the same (and therefore be directed against the sameepitope).

The latter is of particular importance, as it is known that the primarymode of signal transduction by TNF involves crosslinking by TNFreceptors by a trimer of TNF molecules, which contains three receptorbinding sites (see for example Peppel et al., J. Exp. Med., 174 (1991),1483-1489; Engelmann et al., J. Biol. Chem., 265 (1990), 14497; Smithand Baglioni, J. Biol. Chem., 264 (1989), 14646). For example, asdescribed by Peppel et al., an engineered monovalent extracellulardomain of the TNF receptor—which was only capable of blocking a singlereceptor binding site on a TNF trimer—was unable to prevent crosslinkingof the TNF receptors by the remaining two receptor binding sites;whereas an engineered protein that comprises two such extracellulardomains—thus being capable of blocking two receptor bindingsites—provided a striking efficacy compared to the monovalentextracellular domain.

In the present invention, it has been found that monovalent Nanobodiesare capable of binding to TNF alpha in such a way that the activity ofTNF is reduced, both in in vitro models, in cellular models and in exvivo models (see the Experimental Section below). Although the inventionis not limited to any specific mechanism, explanation or hypothesis, itis assumed that because of their small size and high affinity forTNF-alpha, two or three monovalent Nanobodies of the invention arecapable of simultaneously occupying two or three different receptorbinding sites on the TNF trimer, thus preventing the trimer to initiatereceptor crosslinking and thereby to initiate signal transduction(however, other mechanisms of action are not excluded: for example,depending on the epitope against which it is directed, a Nanobody of theinvention may also inhibit the association of TNF into the trimericstate).

It should also be noted that, in addition or as an alternative tobinding to two or more receptor binding sites on a single TNF-trimer,the proteins or polypeptides of the present invention that comprises oressentially consists of two or more immunoglobulin variable domains (orsuitable fragments thereof) that are directed against epitopes ofTNF-alpha may bind (e.g. intermolecularly) epitopes on two separateTNF-alpha molecules (e.g. two separate trimers).

However, according to one particularly preferred embodiment, theinvention relates to a protein or polypeptide that comprises oressentially consists of two or more immunoglobulin variable domains (orsuitable fragments thereof) that are each directed against epitopes onTNF-alpha (and in particular of the TNF-alpha trimer) that lie in and/orform part of the receptor binding site(s) of the TNF trimer, such thatsaid polypeptide, upon binding to a TNF trimer, is capable inhibiting orreducing the TNF receptor crosslinking that is mediated by said TNFtrimer and/or the signal transduction that is mediated by such receptorcrosslinking.

In particular, according to this preferred embodiment, the inventionrelates to a protein polypeptide that comprises or essentially consistof two or more immunoglobulin variable domains (or suitable fragmentsthereof) that are each directed against epitopes on TNF-alpha (and inparticular of the TNF-alpha trimer) that lie in and/or form part of thereceptor binding site(s) of the TNF-trimer, wherein said immunoglobulinvariable domains are linked to each other in such a way that the proteinor polypeptide is capable of simultaneously binding to two or morereceptor binding sites on a single TNF trimer (in other words, iscapable of intramolecular binding to at least two TNF receptor bindingsites on a TNF trimer). In this embodiment, the two or moreimmunoglobulin variable domains are preferably as defined above and aremost preferably Nanobodies (so that the protein or polypeptide is amultivalent Nanobody construct, as further described herein). Also, inthis embodiment, the two or more immunoglobulin variable domains may bethe same or different; and may directed against different epitopeswithin the TNF receptor binding site(s), but are preferably directedagainst the same epitope.

In one preferred aspect of this embodiment, the two or moreimmunoglobulin variable domains are directed against epitopes of theTNF-alpha trimer, which epitopes lie in and/or form part of the TNFreceptor binding site(s). For example, the two or more immunoglobulinvariable domains are preferably directed against and/or can bind to anepitope of the TNF-alpha trimer that comprises the following amino acidresidues: Gln at position 88 and Lys at position 90 on a first TNFmonomer (referred to herein as “monomer A”), and Glu at position 146 ona second TNF monomer (referred to herein as “monomer B”) (in whichMonomer A and Monomer B together, in the TNF trimer, form the TNFreceptor binding site(s)).

As further described below in more details with respect to Nanobodies,in such a protein or polypeptide, the at least two immunoglobulinvariable domains are preferably linked in such a way that the distancebetween the N-terminus and the C-terminus of the two immunoglobulinvariable domains present in such a protein or polypeptide is preferablyat least 50 Angstroms, and more preferably in the region of 55-200Angstroms, and more preferably in the region of Angstroms, and inparticular in the region of 65-150 Angstroms.

In a particularly preferred aspect of this embodiment, these two or moreimmunoglobulin sequences are Nanobodies, and are preferably chosen fromthe Nanobodies described herein. Some particularly preferred Nanobodiesfor use in this embodiment of the invention are PMP1C2 (TNF1, SEQ IDNO:52) and/or PMP5F10 (TNF3, SEQ ID NO: 60), as well as humanized andother variants thereof (as described herein); with PMP1C2 (TNF1, SEQ IDNO:52) and its humanized variants being particularly preferred.

Accordingly, the present embodiment will now be described in more detailwith reference to Nanobodies. However, it will be clear to the skilledperson that the teaching herein may be applied analogously toimmunoglobulin variable domains.

In this embodiment of the invention, the two or more immunoglobulinsequences will usually be linked via one or more suitable linkers, whichlinkers are such that each immunoglobulin sequence can bind to adifferent receptor binding site on the same TNF trimer. Suitable linkerswill inter alia depend on (the distance between) the epitopes on the TNFtrimer to which the immunoglobulin sequences bind, and will be clear tothe skilled person based on the disclosure herein, optionally after somelimited degree of routine experimentation. For example, when the two ormore immunoglobulin sequences are (single) domain antibodies orNanobodies, suitable linkers may be chosen from the linkers describedherein, but with a linker length that is such that the two or more(single) domain antibodies or Nanobodies can each bind to a differentreceptor binding site on the same TNF trimer.

Also, when the two or more immunoglobulin sequences that bind to thereceptor binding sites of TNF-alpha are (single) domain antibodies orNanobodies, they may also be linked to each other via a third (single)domain antibody or Nanobody (in which the two or more immunoglobulinsequences may be linked directly to the third (single) domainantibody/Nanobody or via suitable linkers). Such a third (single) domainantibody or Nanobody may for example be a (single) domain antibody orNanobody that provides for an increased half-life, as further describedherein. For example, the latter (single) domain antibody or Nanobody maybe a (single) domain antibody or Nanobody that is capable of binding toa (human) serum protein such as (human) serum albumin, as furtherdescribed herein.

Alternatively, the two or more immunoglobulin sequences that bind to thereceptor binding site(s) of TNF-alpha may be linked in series (eitherdirectly or via a suitable linker) and the third (single) domainantibody or Nanobody (which may provide for increased half-life, asdescribed above) may be connected directly or via a linker to one ofthese two or more aforementioned immunoglobulin sequences. Somenon-limiting examples of such constructs are the constructs of SEQ IDNOS: 93 or 94.

In particular, it has been found in the invention (see thecrystallography data referred to herein) that, when the Nanobodiespresent in a multivalent or multispecific protein or polypeptide of theinvention bind to the particular epitope described above (which is theepitope of TNF1 and its humanized variants, as well as of TNF3 and itshumanized variants) then preferably, the two (or more) anti-TNFNanobodies present in such a protein or polypeptide should be linked insuch a way that the distance between the N-terminus and the C-terminusof two anti-TNF Nanobodies present in such a protein or polypeptideshould preferably be at least 50 Angstroms, and more preferably in theregion of 55-200 Angstroms, and in particular in the region of 65-150Angstroms (with the upper limit being less critical, and being chosenfor reasons of convenience, e.g. with a view to expression/production ofthe protein); or more generally that said distance should be such thatit allows the protein or polypeptide to undergo intramolecular bindingto the TNF-trimer (i.e. instead of intermolecular binding). The distancebetween the N-terminus and the C-terminus of two anti-TNF Nanobodies canbe determined by any suitable means, such as by crystallography ormolecular modelling (as described herein). These techniques generallyalso make it possible to determine whether a specific multivalent ormultispecific protein or polypeptide is capable of providingintramolecular modelling. Alternatively, the present invention alsoprovides a simple experiment using size-exclusion chromatography (asdescribed by Santora et al., Anal. Biochem., 299: 119-129) that can beused to determine whether a given protein or polypeptide of theinvention will (predominantly) provide intramolecular binding to aTNF-trimer or (predominantly) intermolecular binding between two or moreTNF-trimers. Thus, in one particular embodiment of the invention, aprotein or polypeptide of the invention is preferably such that in thisexperiment, it predominantly or essentially exclusively leads tointramolecular binding However, as emphasized above, it should be notedthat proteins or polypeptides of the invention that operate viaintermolecular binding of separate TNF-alpha molecules (e.g. trimers)are also within the scope of the present invention.

Thus, in another preferred aspect, the invention provides for amultivalent or multispecific protein or polypeptide that comprises atleast two Nanobodies against TNF-alpha (and in particular of theTNF-alpha trimer), in which said Nanobodies are preferably directed toessentially the same epitope as Nanobody PMP1C2 (as mentioned herein),and in which said at least two Nanobodies are linked in such a way thatthe distance the distance between the N-terminus and the C-terminus ofthe at least two anti-TNF Nanobodies is such that the protein orpolypeptide is capable of undergoing intramolecular binding (asdescribed herein) with a TNF-trimer. Preferably, in such a protein orpolypeptide, the distance between the N-terminus and the C-terminus oftwo anti-TNF Nanobodies is at least 50 Angstroms, and more preferably inthe region of 55-200 Angstroms, and in particular in the region of65-150 Angstroms.

In such a preferred protein or polypeptide, the two or more Nanobodiesmay be linked in any suitable fashion, as long as the preferred distancebetween the N-terminus and the C-terminus of the at least two anti-TNFNanobodies can be achieved, and/or as long as the protein or polypeptideis capable of undergoing intramolecular binding (as described herein)with a TNF-trimer.

For example, in its simplest form, the at least two Nanobodies aredirectly linked via a suitable linker or spacer that provides for thepreferred distance between the N-terminus and the C-terminus of the atleast two anti-TNF Nanobodies and which may allow the protein orpolypeptide to undergo intramolecular binding (as described herein) witha TNF-trimer. Suitable linkers are described herein, and may—for exampleand without limitation—comprise an amino acid sequence, which amino acidsequence preferably has a length of 14 amino acids, more preferably atleast 17 amino acids, such as about 20-40 amino acid sequence (which,using an average distance of 3.5 Angstrom for one amino acid,corresponds to linker lengths of 49 Angstroms, 59.5 Angstroms and about70 Angstroms, respectively; with the maximum amount of amino acids beingcalculated in the same way based on the distances mentioned above).Preferably, such an amino acid sequence should also be such that itallows the protein or polypeptide to undergo intramolecular binding (asdescribed herein) with a TNF-trimer.

Thus, in another preferred aspect, the invention provides for amultivalent or multispecific protein or polypeptide that comprises atleast two Nanobodies against TNF-alpha (and in particular of theTNF-alpha trimer), in which said Nanobodies are preferably directed toessentially the same epitope as Nanobody PMP1C2 (as mentioned herein),and in which said at least two Nanobodies are directly linked to eachother using a suitable linker or spacer such that the distance thedistance between the N-terminus and the C-terminus of the at least twoanti-TNF Nanobodies is such that the protein or polypeptide is capableof undergoing intramolecular binding (as described herein) with aTNF-trimer. Preferably, in such a protein or polypeptide, the distancebetween the N-terminus and the C-terminus of two anti-TNF Nanobodies(and thereby the preferred length of the linker or spacer) is at least50 Angstroms, and more preferably in the region of 55-200 Angstroms, andin particular in the region of 65-150 Angstroms.

More preferably, in this preferred aspect, the linker or spacer is anamino acid sequence that comprises at least 14, preferably at least 17,more preferably at least 20 amino acids (with a non-critical upper limitchosen for reasons of convenience being abut 50, and preferably about 40amino acids). In one preferred, but non-limiting embodiment, the linkeressentially consists of glycine and serine residues (as furtherdescribed below). For example, one suitable linker is the GS30 linkerdescribed herein, which comprises 30 amino acid residues.

In another embodiment, the at least two Nanobodies against TNF-alpha arelinked to each other via another moiety (optionally via one or twolinkers), such as another protein or polypeptide. In this embodiment, itmay be desirable to have the preferred distance (i.e. as mentionedabove) between the N-terminus and the C-terminus of the at least twoanti-TNF Nanobodies, for example such that the protein or polypeptidecan still undergo intramolecular binding (as described herein) with aTNF-trimer. In this embodiment, the at least two Nanobodies may belinked directly to the other moiety, or using a suitable linker orspacer, again as long as the preferred distance and/or desiredintramolecular binding can still be achieved. The moiety may be anysuitable moiety which does not detract (too much) from the binding ofthe protein or polypeptide to TNF and/or from the further desiredbiological or pharmacological properties of the protein or polypeptide.As such, the moiety may be essentially inactive or may be biologicallyactive, and as such may or may not improve the desired properties of theprotein or polypeptide and/or may confer one or more additional desiredproperties to the protein or polypeptide. For example, and withoutlimitation, the moiety may improve the half-life of the protein orpolypeptide, and/or may reduce its immunogenicity or improve any otherdesired property. In one preferred embodiment, the moiety may be anotherNanobody (including but not limited to a third Nanobody againstTNF-alpha, although this is not necessary and usually less preferred),and in particular another Nanobody that improves the half-life of theprotein or polypeptide, such as a Nanobody that is directed against aserum protein, for example against human serum albumin. Examples of suchproteins and polypeptides are described herein.

Thus, in one embodiment, the invention relates to a multivalentmultispecific construct comprising two or more immunoglobulin sequences(or suitable fragments thereof) that are each directed against epitopeson TNF-alpha (e.g. of the TNF-alpha trimer) that lie in and/or form partof the receptor binding site, and that are linked to each other via atleast one immunoglobulin sequence that provides for increased half-life(and optionally via one or more suitable linkers), such that saidpolypeptide, upon binding to a TNF trimer, is capable inhibiting orreducing the TNF receptor crosslinking and/or the signal transductionthat is mediated by said TNF trimer. Such a polypeptide may be such suchthat said firstmentioned two or more immunoglobulin sequences can eachbind to a different receptor binding site on a TNF trimer.

In particular, in this embodiment, the polypeptide may comprise atrivalent bispecific Nanobody, that comprises two Nanobodies that areeach directed against epitopes on TNF-alpha (and in particular of theTNF-alpha trimer) that lie in and/or form part of the receptor bindingsite, in which said Nanobodies are linked to each other via a thirdNanobody that provides for an increased half-life (e.g. a Nanobody thatis directed to a serum protein such as human serum albumin), in whicheach of the firstmentioned two Nanobodies may be directly linked to saidthird Nanobody or via one or more suitable linkers, such that saidpolypeptide, upon binding to a TNF trimer, is capable inhibiting orreducing the TNF receptor crosslinking and/or the signal transductionthat is mediated by said TNF trimer. Such a polypeptide may be such thatsaid firstmentioned two Nanobodies can each bind to a different receptorbinding site on a TNF trimer. Again, some particularly preferredNanobodies for use in this embodiment of the invention are PMP1C2 (TNF1,SEQ ID NO:52) and/or PMP5F10 (TNF3, SEQ ID NO: 60), as well as humanizedand other variants thereof (as described herein); with PMP1C2 (TNF1, SEQID NO:52) and its humanized variants being particularly preferred; andthe Nanobodies directed against human serum albumin described herein.Some preferred, but non-limiting constructs of this embodiment of theinvention are TNF 24 (SEQ ID NO: 90), TNF 26 (SEQ ID NO: 92), TNF 27(SEQ ID NO: 93), TNF 28 (SEQ ID NO: 94), TNF 60 (SEQ ID NO: 417) and TNF62 (SEQ ID NO:418), of which TNF 60 (SEQ ID NO: 417) is particularlypreferred.

Thus, some preferred aspects of this embodiment of the invention relateto:

-   XIX) A protein or polypeptide that comprises or essentially consists    of two or more immunoglobulin variable domains (or suitable    fragments thereof) that are each directed against epitopes on    TNF-alpha (and in particular of the TNF-alpha trimer) that lie in    and/or form part of the receptor binding site(s) of the TNF trimer,    such that said polypeptide, upon binding to a TNF trimer, is capable    inhibiting or reducing the TNF receptor crosslinking that is    mediated by said TNF trimer and/or the signal transduction that is    mediated by such receptor crosslinking.-   XX) A protein or polypeptide that comprises or essentially consists    of two or more immunoglobulin variable domains (or suitable    fragments thereof) that are each directed against epitopes on    TNF-alpha (and in particular of the TNF-alpha trimer) that lie in    and/or form part of the receptor binding site(s) of the TNF trimer,    such that said polypeptide is capable intramolecular binding to at    least two TNF receptor binding sites on a TNF trimer.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which said immunoglobulin variable domains are        linked to each other in such a way that the protein or        polypeptide is capable of simultaneously binding to two or more        receptor binding sites on a single TNF trimer.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which said immunoglobulin variable domains are        capable of binding to the same epitope as Nanobody TNF1 (SEQ ID        NO: 52).    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which said immunoglobulin variable domains are        capable of binding against the epitope within the TNF receptor        binding site of the TNF trimer that at least comprises the        following amino acid residues: Gln at position 88 in monomer A;        Lys at position 90 in monomer A and Glu at position 146 in        monomer B.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which said immunoglobulin variable domains are        capable of binding against the epitope within the TNF receptor        binding site of the TNF trimer that at least comprises the        following amino acid residues: Gln at position 88 in monomer A;        Lys at position 90 in monomer A and Glu at position 146 in        monomer B; and that further comprises at least one, preferably        two or more, more preferably 5 or more, and preferably all or        essentially all, of the following amino acid residues of        TNF-alpha monomer A: Gly at position 24, Gln at position 25, Thr        at position 72, His at position 73, Val at position 74, Leu at        position 75, Thr at position 77, Thr at position 79, Be at        position 83, Thr at position 89, Val at position 91. Asn at        position 92, Ile at position 97, Arg at position 131, Glu at        position 135, Ile at position 136, Asn at position 137, Arg at        position 138, Pro at position 139, Asp at position 140 and the        following residues in monomer B: Pro at position 20, Arg at        position 32, Lys at position 65, Lys at position 112, Tyr at        position 115, Ala at position 145, Ser at position 147.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which said immunoglobulin variable domains are        capable of binding against the epitope within the TNF receptor        binding site of the TNF trimer that at least comprises the        following amino acid residues: Gln at position 88 in monomer A;        Lys at position 90 in monomer A and Glu at position 146 in        monomer B; and that further comprises at least one, preferably        two or more, more preferably 5 or more, and preferably all or        essentially all, of the following amino acid residues of        TNF-alpha monomer A Leu at position 75, Thr at position 77, Thr        at position 79, Ile at position 80, Ser at position 81, Tyr at        position 87, Thr at position 89, Val at position 91, Asn at        position 92, Ser at position 95, Be at position 97, Glu at        position 135, Ile at position 136, Asn at position 137 and the        following residues in monomer B: Ala at position 33, Ala at        position 145, Ser at position 147.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the at least two immunoglobulin variable        domains are linked in such a way that the distance between the        N-terminus of the first immunoglobulin variable domain and the        C-terminus of the second immunoglobulin variable domain present        in such a protein or polypeptide is at least 50 Angstroms.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the distance between the N-terminus of        the first immunoglobulin variable domain and the C-terminus of        the second immunoglobulin variable domain is between 55-200        Angstroms    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the distance between the N-terminus of        the first immunoglobulin variable domain and the C-terminus of        the second immunoglobulin variable domain is between 65-150        Angstroms    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domain are linked to each other via a linker or spacer.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the linker or spacer is an amino acid        sequence.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the linker or spacer is comprises at        least 14 amino acid residues.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the linker or spacer is comprises at        least 17-50 amino acid residues.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the linker or spacer essentially consists        of glycine and serine residues.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the linker or spacer is GS30 (SEQ ID NO:        69).    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domain are linked to each other via another moiety.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which said other moiety is a protein or        polypeptide moiety.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which said other moiety confers at least one        desired property to the protein or polypeptide, or improves at        least one desired property of the protein or polypeptide.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which said other moiety improves the half-life        of the protein or polypeptide and/or reduces the immunogenicity        of the protein or polypeptide.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which each of the first and second        immunoglobulin variable domain are linked to said other moiety        via a linker or spacer.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the linker or spacer is an amino acid        sequence.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the linker or spacer essentially consists        of glycine and serine residues.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the said other moiety is a Nanobody.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the other moiety is a Nanobody directed        against human serum albumin.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the at least one Nanobody directed        against human serum albumin is a humanized Nanobody.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the at least one Nanobody directed        against human serum albumin is a humanized variant of the        Nanobody ALB 1 (SEQ ID NO: 63).    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the at least one Nanobody directed        against human serum albumin is a chosen from the group        consisting of ALB 3 (SEQ ID NO: 87), ALB 4 (SEQ ID NO: 88), ALB        5 (SEQ ID NO: 89), ALB 6 (SEQ ID NO: 100), ALB 7 (SEQ ID NO:        101), ALB 8 (SEQ ID NO: 102) ALB 9 (SEQ ID NO: 103) and ALB 10        (SEQ ID NO: 104).    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the at least one Nanobody directed        against human serum albumin is ALB 8.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies with a K_(off) rate for TNF of        better than 2.10⁻³ (1/s), preferably better than 1.10⁻³ (1/s);        or a humanized variant of such a Nanobody.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies with an EC50 value in the        cell-based assay using KYM cells described in Example 1, under        3), of WO 04/041862 that is better than the EC50 value of        Nanobody VHH 3E (SEQ ID NO:4) of WO 04/041862 in the same assay;        or a humanized variant of such a Nanobody.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies with an EC50 value in the        cell-based assay using KYM cells described in Example 1, under        3), of WO 04/041862 that is better than 12 nM; or a humanized        variant of such a Nanobody.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies with an EC50 value in the        cell-based assay using KYM cells described in Example 1, under        3), of WO 04/041862 that is better than 5 nM; or a humanized        variant of such a Nanobody.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies with an EC50 value in the        cell-based assay using KYM cells described in Example 1, under        3), of WO 04/041862 that is better than 3 nM; or a humanized        variant of such a Nanobody.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies in accordance with any one        of XIX) to XX) above, which are humanized;        with some particularly preferred aspects of this embodiment        being:    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are GLEW-class Nanobodies.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies with an arginine residue (R) at        position 103.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are GLEW-class Nanobodies with an arginine        residue (R) at position 103.    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies with at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity (as defined herein) with one of the        amino acid sequences of SEQ ID NO's 52 (TNF1), 76 (TNF13), 77        (TNF14), 95 (TNF29) or 96 (TNF30)    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which:    -   a) CDR1 comprises:        -   the amino acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence DYWMY (SEQ ID NO:            164);    -   and    -   b) CDR2 comprises:        -   the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 233);    -   and    -   c) CDR3 comprises:        -   the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence SPSGFN (SEQ ID            NO: 300).    -   A protein or polypeptide in accordance with any one of XIX)        to XX) above, in which the first and second immunoglobulin        variable domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO: 164).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID        NO: 233).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO: 300)    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which:        -   CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:            164); and CDR3 comprises the amino acid sequence SPSGFN (SEQ            ID NO: 300); or        -   CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:            164); and CDR2 comprises the amino acid sequence            EINTNGLITKYPDSVKG (SEQ ID NO: 233); or        -   CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 233); and CDR3 comprises the amino acid sequence            SPSGFN (SEQ ID NO: 300)    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO: 164);        and CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO:        300).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO: 164);        CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID        NO: 233) and CDR3 comprises the amino acid sequence SPSGFN (SEQ        ID NO: 300).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which    -   a) CDR1 is:        -   the amino acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence DYWMY (SEQ ID NO:            164);    -   and in which:    -   b) CDR2 is:        -   the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 233);    -   and in which    -   c) CDR3 is:        -   the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SPSGFN (SEQ ID NO:            300).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO:        233).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR3 is the amino acid sequence SPSGFN (SEQ ID NO: 300)    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which:        -   CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); and            CDR3 is the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); and            CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID            NO: 233); or        -   CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID            NO: 233); and CDR3 is the amino acid sequence SPSGFN (SEQ ID            NO: 300)    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); and CDR3        is the amino acid sequence SPSGFN (SEQ ID NO: 300).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); CDR2 is        the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO:233) and        CDR3 is the amino acid sequence SPSGFN (SEQ ID NO: 300).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are chosen from the group consisting of the Nanobody TNF        1 (SEQ ID NO: 52) and humanized variants of the Nanobody TNF 1        (SEQ ID NO: 52).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are chosen from the group consisting of TNF 13 (SEQ ID        NO: 76), TNF 14 (SEQ ID NO: 77), TNF 29 (SEQ ID NO: 95) and TNF        30 (SEQ ID NO:96).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are TNF 30 (SEQ ID NO:96);        and with some particularly preferred aspects of this embodiment        being:    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are KERE-class Nanobodies.    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies with at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with one of the amino        acid sequences of SEQ ID NO's 50 (TNF3), 83 (TNF20), 85 (TNF21),        85 (TNF22), 96 (TNF23) or 98 (TNF33).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which:    -   a) CDR1 comprises:        -   the amino acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence NYYMG (SEQ ID NO:            172);    -   and    -   b) CDR2 comprises:        -   the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence NISWRGYNIYYKDSVKG            (SEQ ID NO: 240);    -   and    -   c) CDR3 comprises:        -   the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SILPLSDDPGWNTY (SEQ            ID NO: 308).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO: 172).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR2 comprises the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID        NO: 240).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX) in which        CDR3 comprises the amino acid sequence SILPLSDDPGWNTY (SEQ ID        NO: 308).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX), in        which:        -   CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO:            172); and CDR3 comprises the amino acid sequence            SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO:            172); and CDR2 comprises the amino acid sequence            NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or        -   CDR2 comprises the amino acid sequence NISWRGYNIYYKDSVKG            (SEQ ID NO: 240); and CDR3 comprises the amino acid sequence            SILPLSDDPGWNTY (SEQ ID NO: 308).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX), in which        CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO: 172);        CDR2 comprises the amino acid sequence SILPLSDDPGWNTY (SEQ ID        NO: 308) and CDR3 comprises the amino acid sequence        ILPLSDDPGWNTY (SEQ ID NO: 436).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX), in which    -   a) CDR1 is:        -   the amino acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence NYYMG (SEQ ID NO:            172);    -   and    -   b) CDR2 is:        -   the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence NISWRGYNIYYKDSVKG            (SEQ ID NO: 240);    -   and    -   c) CDR3 is:        -   the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SILPLSDDPGWNTY (SEQ            ID NO: 308).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX), in which        CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX), in which        CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO:        240).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX), in which        CDR3 is the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX), in        which:        -   CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); and            CDR3 is the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO:            308); or        -   CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); and            CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID            NO: 240); or        -   CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID            NO: 240); and CDR3 is the amino acid sequence SILPLSDDPGWNTY            (SEQ ID NO: 308).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX), in which        CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); CDR2 is        the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308) and CDR3        is the amino acid sequence ILPLSDDPGWNTY (SEQ ID NO: 436).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are Nanobodies as described for the proteins or        polypeptides in accordance with any one of XIX) to XX), in which        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are chosen from the group consisting of the Nanobody TNF        3 (SEQ ID NO: 60) and humanized variants of the Nanobody TNF 3        (SEQ ID NO: 60).    -   A protein or polypeptide in accordance with any one of XIX) to        XX), in which the first and second immunoglobulin variable        domains are chosen from the group consisting of TNF20 (SEQ ID        NO:83), TNF21 (SEQ ID NO: 84), TNF 22 (SEQ ID NO: 85), TNF23        (SEQ ID NO: 86) or TNF33 (SEQ ID NO: 99).

It should be noted that when a protein or polypeptide is mentioned aboveas being “in accordance with any one of XIX) to XX) above”, it is atleast according to one of XIX) to XX), and may be according to both XIX)and XX), and may also include any one or more of the other aspects thatare indicated as being “in accordance with any one of XIX) to XX)above”.

However, it should be noted that the invention is not limited to anyspecific mechanism of action or hypothesis. In particular, it has beenfound that the monovalent Nanobodies of the invention may be also activein the assays and models described herein, which confirms thatintramolecular binding of he TNF trimer, although preferred in onespecific embodiment of the invention, is not required to obtain thedesired action and effect of the Nanobodies, proteins and polypeptidesdescribed herein. Similarly, it is also encompassed within the scope ofthe invention that the proteins and polypeptides described hereinachieve their desired action via any appropriate mechanism (i.e. byintramolecular binding, intermolecular binding or even by binding tomonomeric TNF, thus inhibiting the formation of TNF trimers).

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the Nanobodiesand polypeptides of the invention, and/or to use proteins orpolypeptides comprising or essentially consisting of the same, as longas these are suitable for the uses envisaged herein. Such parts,fragments, analogs, mutants, variants, alleles, derivatives, proteinsand/or polypeptides will be described in the further description herein.

In another aspect, the invention relates to a Nanobody (as definedherein), against TNF-alpha, which consist of 4 framework regions (FR1 toFR4 respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which:

-   (i) CDR1 is an amino acid sequence chosen from the group consisting    of the CDR1 sequences of SEQ ID NOS: 15 to 21 or from the group    consisting of the CDR1 sequences of SEQ ID NOS: 164 to 197;    -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with at least one of the CDR1 sequences from the        group consisting of SEQ ID NOS: 15 to 21 or with at least one of        the CDR1 sequences from the group consisting of SEQ ID NOS: 164        to 197, in which    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with at least one of the CDR1 sequences from the group        consisting of SEQ ID NOS: 15 to 21 or with at least one of the        CDR1 sequences from the group consisting of SEQ ID NOS: 164 to        197, in which:    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);    -   and in which:    -   (ii) CDR2 is an amino acid sequence chosen from the group        consisting of the CDR2 sequences of SEQ ID NOS: 22 to 28 or from        the group consisting of the CDR2 sequences of SEQ ID NOS: 232 to        265;        -   or from the group consisting of amino acid sequences that            have at least 80%, preferably at least 90%, more preferably            at least 95%, even more preferably at least 99% sequence            identity (as defined herein) with at least one of the CDR2            sequences from the group consisting of SEQ ID NOS: 22 to 28            or with at least one of the CDR2 sequences from the group            consisting of SEQ ID NOS: 232 to 265, in which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);        -   and/or from the group consisting of amino acid sequences            that have 3, 2 or only 1 “amino acid difference(s)” (as            defined herein) with at least one of the CDR2 sequences from            the group consisting of SEQ ID NOS: 22 to 28 or with at            least one of the CDR2 sequences from the group consisting of            SEQ ID NOS: 232 to 265, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and in which:    -   (iii) CDR3 is an amino acid sequence chosen from the group        consisting of the CDR3 sequences of SEQ ID NOS: 29 to 33 or from        the group consisting of the CDR3 sequences of SEQ ID NOS:        300-333;        -   or from the group consisting of amino acid sequences that            have at least 80%, preferably at least 90%, more preferably            at least 95%, even more preferably at least 99% sequence            identity (as defined herein) with at least one of the CDR3            sequences from the group consisting of SEQ ID NOS: 29 to 33            or with at least one of the CDR3 sequences from the group            consisting of SEQ ID NOS: 300-333, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);        -   and/or from the group consisting of amino acid sequences            that have 3, 2 or only 1 “amino acid difference(s)” (as            defined herein) or with at least one of the CDR3 sequences            from the group consisting of SEQ ID NOS: 300-333, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);        -   or from the group consisting of the CDR3 sequences of SEQ ID            NOS: 34 and 35 or from the group consisting of amino acid            sequences that have at least 80%, preferably at least 90%,            more preferably at least 95%, even more preferably at least            99% sequence identity (as defined herein) with at least one            of the CDR3 sequences of SEQ ID NOS: 34 and 35, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);        -   and/or from the group consisting of amino acid sequences            that have 3, 2 or only 1 “amino acid difference(s)” (as            defined herein) with at least one of the CDR3 sequences of            SEQ ID NOS: 34 and 35, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s).

The Nanobodies against TNF-alpha as described above and as furtherdescribed hereinbelow are also referred to herein as Nanobodies of theinvention.

Of the Nanobodies of the invention, Nanobodies comprising one or more ofthe CDR's explicitly listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's explicitly listed above are moreparticularly preferred; and Nanobodies comprising three of the CDR'sexplicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDRsequences can be seen in Table I below, which lists the CDR's andframework sequences that are present in a number of preferred (butnon-limiting) Nanobodies of the invention. As will be clear to theskilled person, a combination of CDR1, CDR2 and CDR3 sequences thatoccur in the same clone (i.e. CDR1, CDR2 and CDR3 sequences which arementioned on the same line in Table I) will usually be preferred(although the invention in its broadest sense is not limited thereto,and also comprises other suitable combinations of the CDR sequencesmentioned in Table I).

Also, in the Nanobodies of the invention that comprise the combinationsof CDR's mentioned in Table I, each CDR can be replaced by a CDR chosenfrom the group consisting of amino acid sequences that have at least80%, preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with thementioned CDR's; in which

-   -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and/or chosen from the group consisting of amino acid sequences        that have 3, 2 or only 1 (as indicated in the preceding        paragraph) “amino acid difference(s)” (as defined herein) with        the mentioned CDR(s) one of the above amino acid sequences, in        which:    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s).

However, as will be clear to the skilled person, the (combinations of)CDR sequences mentioned in Table I will generally be preferred.

TABLE I Preferred combinations of framework and CDR sequences Clone IDFR1 ID CDR1 ID FR2 ID CDR2 PMP1C2 130 QVQLVESGGGLVQPG 164 DYWMY 198WVRQAPGKG 232 EINTNGLITKYP (TNF1) GSLRLSCAASGFTFS LEWVS DSVKG PMP1G11131 QVQLQESGGGMVQPG 165 VSWMY 199 WVRQAPGKG 233 EINTNGLITKYPGSLRLSCAASGFDFG LEWVS DSVKG PMP1H6 132 EVQLVESGGGLVQPG 166 VSWMY 200WVRQAPGKG 234 EINTNGLITKYV GSLRLSCATSGFDFS LEWVS DSVKG PMP1G5 133QVQLVESGGGLVQAG 167 EPSGYT 201 WFRQAPGKE 235 RIYWSSGLTYY (TNF2)GSLRLSCAASGRTFS YTIG REFVA ADSVKG PMP1H2 134 QVKLEESGGGLVQPG 168 DYSGYT202 WFRQAPGKE 236 RIYWSSGNTYY DSLRLSCAASGRTFS YTVG REFVA ADSVKG PMP3G2135 AVQLVESGGGLVQPG 169 DYSGYT 203 WFRQAPGKE 237 RIYWSSGNTYYDSLRLSCAASGRTFS YTVG REFVA ADSVKG PMP1D2 136 AVQLVDSGGGLVQAG 170 AHSVYT204 WFRQAPGKE 238 RIYWSSANTYY GSLRLSCAASGRTFS MG REFVA ADSVKG PMP3D10137 QVQLVESGGGLVQAGG 171 GYYMG 205 WFRQAPG 239 SISWRGDNTYYKESLSLSCAASGRSFT KERQLLA SVKG PMP5F10 138 EVQLVESGGGLVQAGG 172 NYYMG 206WFRQAPG 240 NISWRGYNIYYKDS (TNF3) SLSLSCSASGRSLS KERELLG VKGNC55TNF_S1C4 139 EVQLVESGGGLVQAGD 173 SHVAA 207 WFRQAPG 241EIRPSGDFGPEGE SLRLSCAASQIIFG REREFVA FEHVTASLKG NC55TNF_S1C3 140EVQLVESGGGLVQPGG 174 AYATG 208 WFRRAPG 242 GIQWSGGDAFYRN SLRLSCKNAGSTSNKEREFVA SVKG NC55TNF_S2C1 141 EVQLVESGGDLVQPGG 175 TNDVG 209 WYRRAPG 243TITDDGTTDYGDD SLRLSCAVSGQLFS KQRELVA VKG NC55TNF_S2C5 142EVQLVESGGGLVQPGG 176 TTSMT 210 WVRQAPG 244 FINSDGSSTTYADS SLRLSCVVSGFTFSKFEEWVS VKG NC55TNF_S3C7 143 EVQLVESGGGTVQAGD 177 SVAMG 211 WFRQAPG 245GVGYDGSSIRYAE SLRLSCAASGRSFS KQREFLA SVKG NC55TNF_S3C1 144 TNF- 178DSAMG 212 WFRQAPGK 246 TISWNGGSS ALPHAVESGGGLMQPG EREFVA SYADFVKGGSLKLSCAASGFMFS NC55TNF_BMP1B2 145 EVQLVESGGGLVQAGG 179 TYAMG 213WFRQAPGK 247 AISWGGGSIV SLRLSCAASGRTFG EREFVA YAESAKG NC55TNF_BMP1D2 146EVQLVESGGELVQAGGS 180 TYAMS 214 WFRRAPGKE 248 SISWSGDTT LKLSCTASGRNFVREFVA YYSNSVKG NC55TNF_BMP1E2 147 EVQLVESGGRLVQPGG 181 AYATG 215WFRRAPGKE 249 GIQWSGGDA SLRLSCKNAGSTSN REFVA FYRNSVKG NC55TNF_BMP1G2 148EVQLVESGGGLVQPGG 182 IVMLG 216 WYRQAPGK 250 SITIGSRTNY SLRLSCAASATISSQREWVA ADSVKG NC55TNF_BMP2A2 149 EVQLVESGGGLVQAGG 183 SYDMG 217 WFRQAPGE251 RISGSDGSTY SLRLSCAASGQTSS GREFVA YSDRAKD NC55TNF_BMP2C2 150EVQLVESGGGLVQPGG 184 TYDMS 218 WVRQAPGK 252 GIDSGGGSP SLRLSCAASGSTFSGLEWVS MYVDSVKG NC55TNF_BMP2F2 151 EVQLVESGGGLVQAG 185 RYNMA 219WFRQAPGK 253 RVDVSGGNT DSLRLSCEASERSSN EREFLA LYGDSVKD NC55TNF_NC10 152EVQLVESGGGLVQPG 186 FSAYSMT 220 WVRQAPGK 254 FINSDGSSTT GSLRLSCVCVSSGCTAEEWVS YADSVNG NC55TNF_NC11 153 EVQLVESGGGLVQAG 187 KNAMG 221 WFRQPPGK255 SIKWNGNNT DSLTLSCASSGRGFY EREFVA YYADSVRG NC55TNF_NC1 154EVQLVESGGGLVQPG 188 ASSMA 222 WVRQAPGK 256 FINSDGSSTT GSLRLSCVFSGFAFSYEEWVS YADSVQG NC55TNF_NC2 155 EVQLVESGGGLVQAG 189 SYAMG 223 WFRQAPGK257 AISWSGTITN GSLRLSCAASGRTFS EREFVA YADSVKG NC55TNF_NC3 156EVQLVESGGGLVQPG 190 ATSMT 224 WVRQAPGK 258 FINSDGSSTT GSLRLSCVVSGFTFSAEEWVS YADSVKG NC55TNF_NC5 157 EVQLVESGGGLVQAG 191 NYDVG 225 WFRQAPGE259 RISGSGDST GSLRLSCAASGGAFS GREIVA YSSNRAKG NC55TNF_NC6 158EVQLVESGGGLVQPGGSL 192 FSAYSMT 226 WVRQAPGKA 260 FINSDGSSTT RLSCECVSSGCTEEFVS YANSVNG NC55TNF_NC7 159 QVQLVESGGGLVQAGGS 193 TADMG 227 WFRQPPGKG261 RISGIDGTTY LRLSCTASGQTSS REFVA YDEPVKG NC55TNF_NC8 160EVQLVESGGGLVQPGGSL 194 TTSMT 228 WVRQAPGKF 262 FINSDGSSTT RLSCVVSGFTFSEEWVS YADSVKG NC55TNF_S2C2 161 EVQLVESGGGLVQPGGSL 195 VNDMG 229WYRQAPGKE 263 TITDDGRTNY RLSCVASASGVK RELVA EDFAKG NC55TNF_S1C6 162EVQLVESGGGLVQAGGSL 196 SVAMG 230 WFRQAPGKE 264 AIGYDGNSIR RLSCAASGRSFGREFVA YGDSVKG NC55TNF_S3C2 163 EVQLVESGGGLVQAGASL 197 DRFNMA 231WFHQAPGKD 265 RIDVAGYNTA RLSCTTSTRTN REFVS YGDFVKG Clone ID FR3 ID CDR3ID FR4 PMP1C2 266 RFTISRDNAKNTLYLQMNSL 300 SPSGFN 334 RGQGT (TNF1)KPEDTALYYCAR QVTVSS PMP1G11 267 RFTISRDNAKTTLYLQMNSL 301 SPSGSF 335RGQGT KPEDTALYYCAR QVTVSS PMP1H6 268 RFTISRDNAKNTLYLQMDSL 302 SPSGSF 336RGQGT IPEDTALYYCAR QVTVSS PMP1G5 269 RFTISRDIAKNTVDLLMNSL 303 RDGIPTSRSV337 WGQGT (TNF2) KPEDTAVYYCAA GSYNY QVTVSS PMP1H2 270RFTISRDIAKNTVDLLMNNL 304 RDGIPTSRSV 338 WGQGT EPEDTAVYYCAA ESYNY QVTVSSPMP3G2 271 RFTISRDIAKNTVDLLMNNL 305 RDGIPTSRSV 339 WGQGT EPEDTAVYYCAAESYNY QVTVSS PMP1D2 272 RFTISRDNAKNTVDLLMNCL 306 RDGIPTSRSV 340 WGQGTKPEDTAVYYCAA EAYNY QVTVSS PMP3D10 273 RFTISRDDAKNTIYLQMN 307 SILPLSDDPG341 WGQG SLKPEDTAVYYCAA WNTN TQVTV SS PMP5F10 274 RFTISRDDAKNTIYLQMN 308SILPLSDDPG 342 WGQG (TNF3) RLKPEDTAVYYCAA WNTY TQVTV SS NC55TNF_S1C4 275RFTIAKNSVDNTVYLQM 309 APYRGGRDYR 343 WGQG NSLKPEDTAVYYCAA WEYEYEY TQVTVSS NC55TNF_S1C3 276 RFRITRDPDNTVYLQMN 310 KLSPYYNDFD 344 WGQGDLKPEDTAIYYCAQ SSNYEY TQVTV SS NC55TNF_S2C1 277 RFVISREGEMVYLEMNS 311NRLRSTWGIR 345 WGQG LKPEDTAVYYCNI YDV TQVTV SS NC55TNF_S2C5 278RFTISRDNAKNTLYLQMN 312 RGYGRD 346 RSKGI SLKPEDTAMYYCGR QVTVASNC55TNF_S3C7 279 RFTIARGNRESTVFLQM 313 EPIGAYEGLW 347 WGQGENLKPEDTAVYFCTA TY TQVTV SS NC55TNF_S3C1 280 RFTISRDNAKNTVYLQMNG 314SYSNGNPHRFS 348 WGQG LTPQDTAIYYCAG QYQY TQVTV SS NC55TNF_BMP1B2 281RFTISRDNAKXTMYLQMDS 315 ANNIATLRQGS 349 WGQG LKPEDTAVYYCAA TQVTV SSNC55TNF_BMP1D2 282 RFTVSRDNGKNTAYLRMN 316 VQVIDPSWSGV 350 LGSGTSLKPEDTADYYCAV NLDDYDY QVTVSS NC55TNF_BMP1E2 283 RFRITRDPDNTVYLQMNDL 317KLSPYYNDFDS 351 WGQG KPEDTAIYYCAQ SNYEY TQVTV SS NC55TNF_BMP1G2 284RFTISRDNAKNTVYLQMNS 318 VPPRDDY 352 WGQG LKPEDTAVYFCNA TQVTV SSNC55TNF_BMP2A2 285 RFTISRDNTKNMVYLQMD 319 PRYENQWSSY 353 WGQGRLKPDDTAVYYCRV DY TQVTV SS NC55TNF_BMP2C2 286 RFTVSRDNAKNTLYLQMN 320FSTGADGGSW 354 WGKGT SLKPEDTAVYYCAK YWSYGMDS QVTVSS NC55TNF_BMP2F2 287RFTVSRINGKNAMYLQMNNL 321 GGWGTTQYDY 355 WGQG KPEDTAIYYCAA DY TQVTV SSNC55TNF_NC10 288 RFKISRDNAKKTLYLQMNSL 322 RGYALD 356 RGQGT GPEDTAMYYCQRQVTVSS NC55TNF_NC11 289 RFTISRGNAKNTENTVSLQM 323 DSSHYSYVYSK 357 WGQGNSLKPEDTADYYCAA AYEYDY TQVTV SS NC55TNF_NC1 290 RFTISRDNAKNTLYLQMNSL 324RGYGRD 358 RSQGI KSEDTAMYYCGR QVTVSS NC55TNF_NC2 291RFTISRDNGKNTVHLQMNSL 325 VQPYSGGDYY 359 WGXG KPEDTAVYHCAV TGVEEYDY TQVTVSS NC55TNF_NC3 292 RFTISRDNAKNTLYLQMDDL 326 RGYGRD 360 RSRGIQSEDTAMYYCGR QVTVSS NC55TNF_NC5 293 RFTISRDNAKNTVYLQMNSL 327 ARYNGTWSSN361 WGQG KREDTAVYYCRA DY TQVTV SS NC55TNF_NC6 294 RFKISRDNAKKTLYLQMNSL328 RGYALD 362 RGQGTQ GPEDTAMYYCQR VTVSS NC55TNF_NC7 295RFTISRDKAQNTVYLQMDSL 329 PRYADQW 363 WGQGTQ KPEDTAVYYCRS SAYDY VTVSSNC55TNF_NC8 296 RFTISRDNAKNTLYLQMNSL 330 RGYGRD 364 RSKGIQV KPEDTAMYYCGRTVSS NC55TNF_S2C2 297 RFTISRDNAKNTVYLQMNSL 331 RTYWAHL 365 WGQGTQLPEDTAVYYCNA PTY VTVSS NC55TNF_S1C6 298 RFTISRDNIKNTMYLEMENL 332 EPLARYE366 WGQGTQ NADDTARYLCAA GLWTY VTVSS NC55TNF_S3C2 299RFTVSRDSAENTVVLQMNSL 333 GGWGISQ 367 WGQGTQ RPEDTGVYYCAA SDYDL VTVSSNotes to Table I: ID refers to the SEQ ID NO's in the attached sequencelisting For CDR1: SEQ ID NO: 164 corresponds to SEQ ID NO: 15, SEQ IDNO: 167 corresponds to SEQ ID NO: 16; SEQ ID NO: 172 corresponds to SEQID NO: 17, SEQ ID NOS: 165 and 166 correspond to SEQ ID NO: 18, SEQ IDNO: 170 corresponds to SEQ ID NO: 19, SEQ ID NO: 171 corresponds to SEQID NO: 20, and SEQ ID NOS: 168 and 169 correspond to SEQ ID NO: 21. ForCDR2: SEQ ID NOS: 232 and 233 correspond to SEQ ID NO: 22, SEQ ID NO:235 corresponds to SEQ ID NO: 23, SEQ ID NO: 240 corresponds to SEQ IDNO: 24, SEQ ID NO: 234 corresponds to SEQ ID NO: 25, SEQ ID NOS: 236 and237 correspond to SEQ ID NO: 26, SEQ ID NO: 238 corresponds to SEQ IDNO: 27, and SEQ ID NO: 239 corresponds to SEQ ID NO: 28. For CDR3: SEQID NO: 303 corresponds to SEQ ID NO: 29, SEQ ID NO: 308 corresponds toSEQ ID NO: 30, SEQ ID NO: 306 corresponds to SEQ ID NO: 31, SEQ ID NO:307 corresponds to SEQ ID NO: 32, SEQ ID NOS: 304 and 305 corresponds toSEQ ID NO: 33, SEQ ID NO: 300 corresponds to SEQ ID NO: 34, and SEQ IDNOS: 301 and 302 correspond to SEQ ID NO: 35.

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2and CDR3 sequences present is suitably chosen from the group consistingof the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table I;or from the group of CDR1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% “sequence identity” (as definedherein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table I; and/or from the group consisting of theCDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1“amino acid difference(s)” (as defined herein) with at least one of theCDR1, CDR2 and CDR3 sequences, respectively, listed in Table I. In thiscontext, by “suitably chosen” is meant that, as applicable, a CDR1sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein) and a CDR3 sequence is chosen from suitable CDR3sequences (i.e. as defined herein), respectively.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table I or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table I; and/or from the groupconsisting of the CDR3 sequences that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR3 sequences listed in Table I.

Preferably, in the Nanobodies of the invention, at least two of theCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable I or from the group consisting of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table I; and/or from the group consisting of theCDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1“amino acid difference(s)” with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table I.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table I or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table I, respectively; and atleast one of the CDR1 and CDR2 sequences present is suitably chosen fromthe group consisting of the CDR1 and CDR2 sequences, respectively,listed in Table I or from the group of CDR1 and CDR2 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table I; and/or from the group consisting of the CDR1 and CDR2sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table I.

Most preferably, in the Nanobodies of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable I or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table I; and/or from the group consisting of theCDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table I.

Even more preferably, in the Nanobodies of the invention, at least oneof the CDR1, CDR2 and CDR3 sequences present is suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table I. Preferably, in this embodiment, at least one orpreferably both of the other two CDR sequences present are suitablychosen from CDR sequences that that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% sequence identity with at least one of the corresponding CDRsequences, respectively, listed in Table I; and/or from the groupconsisting of the CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with at least one of the corresponding sequences,respectively, listed in Table I.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table I. Preferably, in this embodiment, at least one andpreferably both of the CDR1 and CDR2 sequences present are suitablychosen from the groups of CDR1 and CDR2 sequences, respectively, thatthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with theCDR1 and CDR2 sequences, respectively, listed in listed in Table I;and/or from the group consisting of the CDR1 and CDR2 sequences,respectively, that have 3, 2 or only 1 amino acid difference(s) with atleast one of the CDR1 and CDR2 sequences, respectively, listed in TableI.

Even more preferably, in the Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table I. Preferably, in this embodiment, the remaining CDRsequence present are suitably chosen from the group of CDR sequencesthat that have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the corresponding CDR sequences listed in Table I; and/orfrom the group consisting of CDR sequences that have 3, 2 or only 1amino acid difference(s) with at least one of the correspondingsequences listed in Table I.

In particular, in the Nanobodies of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the CDR3sequences listed in Table I, and either the CDR1 sequence or the CDR2sequence is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table I. Preferably, in thisembodiment, the remaining CDR sequence present are suitably chosen fromthe group of CDR sequences that that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% sequence identity with at least one of the corresponding CDRsequences listed in Table I; and/or from the group consisting of CDRsequences that have 3, 2 or only 1 amino acid difference(s) with thecorresponding CDR sequences listed in Table I.

Even more preferably, in the Nanobodies of the invention, all threeCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable I.

Also, generally, the combinations of CDR's listed in Table I (i.e. thosementioned on the same line in Table I) are preferred. Thus, it isgenerally preferred that, when a CDR in a Nanobody of the invention is aCDR sequence mentioned in Table I or is suitably chosen from the groupof CDR sequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table I; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table I, that at least oneand preferably both of the other CDR's are suitably chosen from the CDRsequences that belong to the same combination in Table I (i.e. mentionedon the same line in Table I) or are suitably chosen from the group ofCDR sequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDR's mentioned in Table I.

Thus, by means of non-limiting examples, a Nanobody of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table I, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table I (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table I; a CDR2 sequence that has 3, 2 or 1amino acid difference with one of the CDR2 sequences mentioned in TableI (but belonging to a different combination); and a CDR3 sequence thathas more than 80% sequence identity with one of the CDR3 sequencesmentioned in Table I (but belonging to a different combination); or (2)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table I; a CDR2 sequence, and one of theCDR3 sequences listed in Table I; or (3) a CDR1 sequence; a CDR2sequence that has more than 80% sequence identity with one of the CDR2sequence listed in Table I; and a CDR3 sequence that has 3, 2 or 1 aminoacid differences with the CDR3 sequence mentioned in Table I thatbelongs to the same combination as the CDR2 sequence.

Some particularly preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table I; a CDR2 sequencethat has 3, 2 or 1 amino acid difference with the CDR2 sequencementioned in Table I that belongs to the same combination; and a CDR3sequence that has more than 80% sequence identity with the CDR3 sequencementioned in Table I that belongs to the same combination; (2) a CDR1sequence; a CDR2 listed in Table I and a CDR3 sequence listed in Table I(in which the CDR2 sequence and CDR3 sequence may belong to differentcombinations).

Some even more preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table I; the CDR2 sequencelisted in Table I that belongs to the same combination; and a CDR3sequence mentioned in Table I that belongs to a different combination;or (2) a CDR1 sequence mentioned in Table I; a CDR2 sequence that has 3,2 or 1 amino acid differences with the CDR2 sequence mentioned in TableI that belongs to the same combination; and more than 80% sequenceidentity with the CDR3 sequence listed in Table I that belongs to samedifferent combination.

Particularly preferred Nanobodies of the invention may for examplecomprise a CDR1 sequence mentioned in Table I, a CDR2 sequence that hasmore than 80% sequence identity with the CDR2 sequence mentioned inTable I that belongs to the same combination; and the CDR3 sequencementioned in Table I that belongs to the same.

In the most preferred in the Nanobodies of the invention, the CDR1, CDR2and CDR3 sequences present are suitably chosen from the one of thecombinations of CDR1, CDR2 and CDR3 sequences, respectively, listed inTable I.

Preferably, when a CDR sequence is suitably chosen from the group of CDRsequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity (as defined herein) with one of the CDR sequences listed inTable I; and/or when a CDR sequence is suitably chosen from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with one of the CDR sequences listed in Table I:

-   -   i) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the CDR sequence listed in Table I.

According to a non-limiting but preferred embodiment of the invention,the CDR sequences in the Nanobodies of the invention are as definedabove and are also such that the Nanobody of the invention binds toTNF-alpha with an dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²moles/liter (M) or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter (M) orless and more preferably 10⁻⁸ to 10⁻¹² moles/liter (M), and/or with anassociation constant (K_(A)) of at least 10⁷ M⁻¹, preferably at least10⁸ M⁻¹, more preferably at least 10⁹ M⁻¹, such as at least 10¹² M⁻¹;and in particular with a K_(D) less than 500 nM, preferably less than200 nM, more preferably less than 10 nM, such as less than 500 pM. TheK_(D) and K_(A) values of the Nanobody of the invention againstTNF-alpha can be determined in a manner known per se, for example usingthe assay described herein.

According to another preferred, but non-limiting embodiment of theinvention (a) CDR1 has a length of between 1 and 12 amino acid residues,and usually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In one aspect, the invention provides Nanobodies against TNF-alpha thatare better performing than Nanobody 3E, the best performing Nanobodyaccording to WO 04/041862.

More specifically, some preferred aspects of this embodiment of theinvention are:

-   XXI) A Nanobody against TNF-alpha, which consist of 4 framework    regions (FR1 to FR4 respectively) and 3 complementarity determining    regions (CDR1 to CDR3 respectively), which has a Koff rate for TNF    of better than 2.10-3 (1/s), preferably better than 1.10-3 (1/s); or    a humanized variant of such a Nanobody.-   XXII) A Nanobody against TNF-alpha, which consist of 4 framework    regions (FR1 to FR4 respectively) and 3 complementarity determining    regions (CDR1 to CDR3 respectively), which has an EC50 value in the    cell-based assay using KYM cells described in Example 1, under 3),    of WO 04/041862 that is better than the EC50 value of Nanobody VHH    3E (SEQ ID NO:4) of WO 04/041862 in the same assay; or a humanized    variant of such a Nanobody.-   XXIII) A Nanobody against TNF-alpha, which has an EC50 value in the    cell-based assay using KYM cells described in Example 1, under 3),    of WO 04/041862 that is better than 12 nM; or a humanized variant of    such a Nanobody.-   XXIV) A Nanobody against TNF-alpha, which has an EC50 value in the    cell-based assay using KYM cells described in Example 1, under 3),    of WO 04/041862 that is better than 5 nM; or a humanized variant of    such a Nanobody.-   XXV) A Nanobody against TNF-alpha, which has an EC50 value in the    cell-based assay using KYM cells described in Example 1, under 3),    of WO 04/041862 that is better than 3 nM; or a humanized variant of    such a Nanobody;    with some particularly preferred aspects being:    -   A Nanobody in accordance with any one of XXI) to XXV), which is        a GLEW-class Nanobody.    -   A Nanobody in accordance with any one of XXI) to XXV), which        contains an arginine residue (R) at position 103.    -   A Nanobody in accordance with any one of XXI) to XXV), which is        a humanized Nanobody.    -   A Nanobody in accordance with any one of XXI) to XXV), which        contains a leucine residue (L) at position 108.    -   A Nanobody in accordance with any one of XXI) to XXV), which has        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the amino acid sequences of SEQ ID        NO's 52 (TNF1), 76 (TNF13), 77 (TNF14), 95 (TNF29) or 96        (TNF30).    -   A Nanobody in accordance with any one of XXI) to XXV), in which    -   a) CDR1 comprises:        -   the amino acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence DYWMY (SEQ ID NO:            164);    -   and    -   b) CDR2 comprises:        -   the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 233);    -   and    -   c) CDR3 comprises:        -   the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence SPSGFN (SEQ ID            NO: 300).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO 164).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID        NO: 233).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO: 300)    -   A Nanobody in accordance with any one of XXI) to XXV), in which:        -   CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:            164); and CDR3 comprises the amino acid sequence SPSGFN (SEQ            ID NO: 300); or        -   CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:            164); and CDR2 comprises the amino acid sequence            EINTNGLITKYPDSVKG (SEQ ID NO: 233); or        -   CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 233); and CDR3 comprises the amino acid sequence            SPSGFN (SEQ ID NO: 300)    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO: 164);        and CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO:        300).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO: 164);        CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID        NO: 233) and CDR3 comprises the amino acid sequence SPSGFN (SEQ        ID NO: 300).    -   A Nanobody in accordance with any one of XXI) to XXV), in which    -   a) CDR1 is:        -   the amino acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence DYWMY (SEQ ID NO:            164);    -   and in which:    -   b) CDR2 is:        -   the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 233);    -   and in which    -   c) CDR3 is:        -   the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SPSGFN (SEQ ID NO:            300).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO:        233).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR3 is the amino acid sequence SPSGFN (SEQ ID NO: 300)    -   A Nanobody in accordance with any one of XXI) to XXV), in which:        -   CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); and            CDR3 is the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164; and            CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID            NO: 233); or        -   CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID            NO: 233); and CDR3 is the amino acid sequence SPSGFN (SEQ ID            NO: 300)    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); and CDR3        is the amino acid sequence SPSGFN (SEQ ID NO: 300).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); CDR2 is        the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233) and        CDR3 is the amino acid sequence SPSGFN (SEQ ID NO: 300).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).            and with some other particularly preferred aspects being:    -   A Nanobody in accordance with any one of XXI) to XXV), which is        a KERE-class Nanobody.    -   A Nanobody in accordance with any one of XXI) to XXV), which has        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the amino acid sequences of SEQ ID        NO's 50 (TNF3), 83 (TNF20), 85 (TNF21), 85 (TNF22), 96 (TNF23)        or 98 (TNF33).    -   A Nanobody in accordance with any one of XXI) to XXV), in which    -   a) CDR1 comprises:        -   the amino acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence NYYMG (SEQ ID NO:            172);    -   and    -   b) CDR2 comprises:        -   the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence NISWRGYNIYYKDSVKG            (SEQ ID NO: 240);    -   and    -   c) CDR3 comprises:        -   the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SILPLSDDPGWNTY (SEQ            ID NO: 308).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO: 172).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR2 comprises the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID        NO: 240).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR3 comprises the amino acid sequence SILPLSDDPGWNTY (SEQ ID        NO: 308).    -   A Nanobody in accordance with any one of XXI) to XXV), in which:        -   CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO:            172); and CDR3 comprises the amino acid sequence            SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO:            172); and CDR2 comprises the amino acid sequence            NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or        -   CDR2 comprises the amino acid sequence NISWRGYNIYYKDSVKG            (SEQ ID NO: 240); and CDR3 comprises the amino acid sequence            SILPLSDDPGWNTY (SEQ ID NO: 308).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO: 172);        CDR2 comprises the amino acid sequence SILPLSDDPGWNTY (SEQ ID        NO: 308) and CDR3 comprises the amino acid sequence        ILPLSDDPGWNTY (SEQ ID NO: 436).    -   A Nanobody in accordance with any one of XXI) to XXV), in which    -   a) CDR1 is:        -   the amino acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence NYYMG (SEQ ID NO:            172);    -   and    -   b) CDR2 is:        -   the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence NISWRGYNIYYKDSVKG            (SEQ ID NO: 240);    -   and    -   c) CDR3 is:        -   the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SILPLSDDPGWNTY (SEQ            ID NO: 308).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO:        240).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR3 is the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308).    -   A Nanobody in accordance with any one of XXI) to XXV), in which:        -   CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); and            CDR3 is the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO:            308); or        -   CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); and            CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID            NO: 240); or        -   CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID            NO: 240); and CDR3 is the amino acid sequence SILPLSDDPGWNTY            (SEQ ID NO: 308).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); CDR2 is        the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308) and CDR3        is the amino acid sequence ILPLSDDPGWNTY (SEQ ID NO: 436).    -   A Nanobody in accordance with any one of XXI) to XXV), in which        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).    -   A Nanobody in accordance with any one of XXI) to XXV), which is        a humanized Nanobody.        and with yet some other particularly preferred aspects being:-   XXVI) A protein or polypeptide, which comprises or essentially    consists of at least one Nanobody in accordance with any one of XXI)    to XXV).-   XXVII) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of XXI) to XXV).-   XXVIII) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of XXI) to XXV), and which is such that said    protein or polypeptide, upon binding to a TNF trimer, is capable    inhibiting or reducing the TNF receptor crosslinking that is    mediated by said TNF trimer and/or the signal transduction that is    mediated by such receptor crosslinking.-   XXIX) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of XXI) to XXV), and which is capable of    intramolecular binding to at least two TNF receptor binding sites on    a TNF trimer.-   XXX) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of XXI) to XXV), linked via a suitable    linker.-   XXXI) A protein or polypeptide, which comprises two Nanobodies in    accordance with any one of XXI) to XXV), linked via a suitable    linker, and which is pegylated.-   XXXII) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of XXI) to XXV), and which further comprises    at least one Nanobody directed against human serum albumin.-   XXXIII) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of XXI) to XXV), and which further comprises    at least one Nanobody directed against human serum albumin, and    which is such that said protein or polypeptide, upon binding to a    TNF trimer, is capable inhibiting or reducing the TNF receptor    crosslinking that is mediated by said TNF trimer and/or the signal    transduction that is mediated by such receptor crosslinking.-   XXXIV) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of XXI) to XXV), and which further comprises    at least one Nanobody directed against human serum albumin and which    is capable of intramolecular binding to at least two TNF receptor    binding sites on a TNF trimer.-   XXXV) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of XXI) to XXV), and which further comprises    one Nanobody directed against human serum albumin, in which each of    the two Nanobodies in accordance with any one of XXI) to XXV) is    linked, optionally via a suitable linker, to the one Nanobody    directed against human serum albumin.-   XXXVI) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of XXI) to XXV), and which further comprises    one Nanobody directed against human serum albumin, in which each of    the two Nanobodies in accordance with any one of XXI) to XXV) is    linked, optionally via a suitable linker, to the one Nanobody    directed against human serum albumin, and which is such that said    protein or polypeptide, upon binding to a TNF trimer, is capable    inhibiting or reducing the TNF receptor crosslinking that is    mediated by said TNF trimer and/or the signal transduction that is    mediated by such receptor crosslinking.-   XXXVII) A protein or polypeptide which comprises two Nanobodies in    accordance with any one of XXI) to XXV), and which further comprises    one Nanobody directed against human serum albumin, in which each of    the two Nanobodies in accordance with any one of XXI) to XXV) is    linked, optionally via a suitable linker, to the one Nanobody    directed against human serum albumin, and which is capable of    intramolecular binding to at least two TNF receptor binding sites on    a TNF trimer.    -   A protein or polypeptide in accordance with any one of XXVI) to        XXXVII), in which the at least one Nanobody directed against        human serum albumin is a humanized Nanobody.    -   A protein or polypeptide in accordance with any one of XXVI) to        XXXVII), in which the at least one Nanobody directed against        human serum albumin is a humanized variant of the Nanobody ALB 1        (SEQ ID NO: 63).    -   A protein or polypeptide in accordance with any one of XXVI) to        XXXVII), in which the at least one Nanobody directed against        human serum albumin is a chosen from the group consisting of ALB        3 (SEQ ID NO: 87), ALB 4 (SEQ ID NO: 88), ALB 5 (SEQ ID NO: 89),        ALB 6 (SEQ ID NO: 100), ALB 7 (SEQ ID NO: 101), ALB 8 (SEQ ID        NO: 102) ALB 9 (SEQ ID NO: 103) and ALB 10 (SEQ ID NO: 104).    -   A protein or polypeptide in accordance with any one of XXVI) to        XXXVII), in which the at least one Nanobody directed against        human serum albumin is ALB 8.    -   A protein or polypeptide in accordance with any one of XXVI) to        XXXVII), which comprises or essentially consists of two        humanized Nanobodies Nanobodies in accordance with any one        of XXI) to XXV), and one humanized variant of the Nanobody ALB 1        (SEQ ID NO: 63).

It should be noted that when a Nanobody is mentioned above as being “inaccordance with any one of XXI) to XXV) above”, it is at least accordingto one of XXI) to XXV), may be according to two or more of XXI) to XXV),and may also include any one or more of the other aspects that areindicated as being “in accordance with any one of XXI) to XXV) above.Similarly, when a protein or polypeptide is mentioned above as being “inaccordance with any one of XXVI) to XXXVII) above”, it is at leastaccording to one of XXVI) to XXXVII), may be according to two or more ofXXVI) to XXXVII), and may also include any one or more of the otheraspects that are indicated as being “in accordance with any one of XXVI)to XXXVII) above.

A clone that has been found to be particularly useful as an anti-TNFNanobody is the clone PMP1C2 (TNF1). As can be seen from the comparativedata from the KYM-assay in Table 39, TNF1 has an EC50 value that is morethen 4 times better than the best monovalent Nanobody described in WO04/41862 (Nanobody 3E), i.e. 2,466 nM for PMP1C2 vs. 12 nM for 3E (Ascan be seen from Table 39, all Nanobodies TNF1 to TNF 9 of the inventiongave a better EC50 value in this assay than 3E). In this respect, itshould also be noted that Nanobody 3E from WO 04/41862 belongs to the“KERE class” (as described herein), and can therefore be humanized to alesser degree than Nanobody PMP1C2 (which belongs to the “GLEW class”).When Nanobody PMP1C2 is compared to the Nanobody 1A from WO 04/41862, aGLEW-class Nanobody with the highest degree in sequence homology withPMP1C2 (in both the CDR's and the frameworks), the EC₅₀ value obtainedfor PMP1C2 in the KYM assay is more than 50 times better, i.e. 2.466 nMfor PMP1C2 compared to 100 nM for 1A.

Accordingly, Nanobodies that comprise one or more, preferably any twoand more preferably all three of the CDR's present in the clone PMP1C2(or CDR sequences that are derived therefrom or correspond thereto) areparticularly preferred in the invention. Also, these Nanobodiespreferably belong to the “103 P,R,S group” (as defined herein), and mostpreferably have an R at position 103, and preferably also have GLEW or aGLEW-like sequence at positions 44-47. Also, when these Nanobodiesbelong to the “103 P, R, S group” (and in particular when they have an Rat position 103), one preferred, but non-limiting humanizingsubstitution is 108Q to 108L. Other preferred, but non-limitinghumanizing substitutions in these preferred Nanobodies are one or moreof those present in the humanized variants of TNF1 described herein,such as TNF13, TNF14, TNF 29 or TNF30, as will immediately be clear froma comparison between the sequence of TNF1 and these humanized sequences.

Thus, in a particularly preferred Nanobody of the invention, at leastone of the CDR1, CDR2 and CDR3 sequences present is suitably chosen fromthe group consisting of the CDR1 sequence of SEQ ID NO: 164, the CDR2sequence of SEQ ID NO: 232, and the CDR3 sequence of SEQ ID NO: 300,respectively (i.e. the CDR sequences present in clone TNF1), or from thegroup of CDR1, CDR2 and CDR3 sequences, respectively, that have at least80%, preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% “sequence identity” (as defined herein) with theCDR1 sequence of SEQ ID NO: 164, the CDR2 sequence of SEQ ID NO: 232,and the CDR3 sequence of SEQ ID NO: 300, respectively; and/or from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,that have 3, 2 or only 1 “amino acid difference(s)” (as defined herein)with the CDR1 sequence of SEQ ID NO: 164, the CDR2 sequence of SEQ IDNO: 232, and the CDR3 sequence of SEQ ID NO: 300, respectively.

Preferably, in these preferred Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1 sequence of SEQ ID NO: 164, the CDR2sequence of SEQ ID NO: 232, and the CDR3 sequence of SEQ ID NO: 300,respectively (i.e. the CDR sequences present in clone TNF1), or from thegroup consisting of CDR1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with the CDR1sequence of SEQ ID NO: 164, the CDR2 sequence of SEQ ID NO: 232, and theCDR3 sequence of SEQ ID NO: 300, respectively; and/or from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have3, 2 or only 1 “amino acid difference(s)” with the CDR1 sequence of SEQID NO: 164, the CDR2 sequence of SEQ ID NO: 232, and the CDR3 sequenceof SEQ ID NO: 300, respectively.

Most preferably, in these preferred Nanobodies of the invention, allthree CDR1, CDR2 and CDR3 sequences present are suitably chosen from thegroup consisting of the CDR1 sequence of SEQ ID NO: 164, the CDR2sequence of SEQ ID NO: 232, and the CDR3 sequence of SEQ ID NO: 300,respectively (i.e. the CDR sequences present in clone TNF1), or from thegroup of CDR1, CDR2 and CDR3 sequences, respectively, that have at least80%, preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with the CDR1 sequence of SEQID NO: 164, the CDR2 sequence of SEQ ID NO: 232, and the CDR3 sequenceof SEQ ID NO: 300, respectively; and/or from the group consisting of theCDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1amino acid difference(s) with the CDR1 sequence of SEQ ID NO: 164, theCDR2 sequence of SEQ ID NO: 232, and the CDR3 sequence of SEQ ID NO:300, respectively.

Even more preferably, in these preferred Nanobodies of the invention, atleast one of the CDR1, CDR2 and CDR3 sequences present is suitablychosen from the group consisting of the CDR1 sequence of SEQ ID NO: 164,the CDR2 sequence of SEQ ID NO: 232, and the CDR3 sequence of SEQ ID NO:300, respectively (i.e. the CDR sequences present in clone TNF1).Preferably, in this embodiment, at least one or preferably both of theother two CDR sequences present are suitably suitably chosen from CDRsequences that that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR1 sequence of SEQ ID NO: 164, the CDR2 sequence ofSEQ ID NO: 232, and the CDR3 sequence of SEQ ID NO: 300, respectively;and/or suitably chosen from the group consisting of the CDR sequencesthat have 3, 2 or only 1 amino acid difference(s) with the CDR1 sequenceof SEQ ID NO: 164, the CDR2 sequence of SEQ ID NO: 232, and the CDR3sequence of SEQ ID NO: 300, respectively.

Even more preferably, in these preferred Nanobodies of the invention, atleast two of the CDR1, CDR2 and CDR3 sequences present are suitablychosen from the group consisting of the CDR1 sequence of SEQ ID NO: 164,the CDR2 sequence of SEQ ID NO: 232, and the CDR3 sequence of SEQ ID NO:300, respectively (i.e. the CDR sequences present in clone TNF1).Preferably, in this embodiment, the remaining CDR sequence present aresuitably chosen from the group of CDR sequences that that have at least80%, preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with the CDR1 sequence of SEQID NO: 164, the CDR2 sequence of SEQ ID NO: 232, and the CDR3 sequenceof SEQ ID NO: 300, respectively; and/or from the group consisting of CDRsequences that have 3, 2 or only 1 amino acid difference(s) with theCDR1 sequence of SEQ ID NO: 164, the CDR2 sequence of SEQ ID NO: 232,and the CDR3 sequence of SEQ ID NO: 300, respectively.

Particularly preferred Nanobodies of the invention comprise the CDR1sequence of SEQ ID NO: 164, the CDR2 sequence of SEQ ID NO: 232, and theCDR3 sequence of SEQ ID NO: 300, respectively (i.e. the CDR sequencespresent in clone TNF1).

Nanobodies with the above CDR sequences preferably have frameworksequences that are as further defined herein. Some particularlypreferred, but non-limiting combinations of framework sequences can beseen in the above Table I. As will be clear to the skilled person, acombination of FR1, FR2, FR3 and FR4 sequences that occur in the sameclone (i.e. FR1, FR2, FR3 and FR4 sequences which are mentioned on thesame line in Table I) will usually be preferred (although the inventionin its broadest sense is not limited thereto, and also comprises othersuitable combinations of the framework sequences mentioned in Table I).

More specifically, some preferred aspects of this embodiment of theinvention are:

-   XXXVIII) A nanobody against TNF-alpha, which consist of 4 framework    regions (FR1 to FR4 respectively) and 3 complementarity determining    regions (CDR1 to CDR3 respectively), in which:    -   a) CDR1 comprises:        -   the amino acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequences that has only 1 amino acid            difference with the amino acid sequence DYWMY (SEQ ID NO:            164);    -   and    -   b) CDR2 comprises:        -   the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 233);    -   and    -   c) CDR3 comprises:        -   the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequences that has only 1 amino acid            difference with the amino acid sequence SPSGFN (SEQ ID NO:            300).    -   A nanobody in accordance with XXXVIII), in which CDR1 comprises        the amino acid sequence DYWMY (SEQ ID NO: 164).    -   A nanobody in accordance with XXXVIII), in which CDR2 comprises        the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233).    -   A nanobody in accordance with XXXVIII), in which CDR3 comprises        the amino acid sequence SPSGFN (SEQ ID NO: 300).    -   A nanobody in accordance with XXXVIII), in which:        -   CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:            164); and CDR3 comprises the amino acid sequence SPSGFN (SEQ            ID NO: 300); or        -   CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO:            164); and CDR2 comprises the amino acid sequence            EINTNGLITKYPDSVKG (SEQ ID NO: 233); or        -   CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 233); and CDR3 comprises the amino acid sequence            SPSGFN (SEQ ID NO: 300)    -   A nanobody in accordance with XXXVIII), in which CDR1 comprises        the amino acid sequence DYWMY (SEQ ID NO: 164); and CDR3        comprises the amino acid sequence SPSGFN (SEQ ID NO: 300).    -   A nanobody in accordance with XXXVIII) in which CDR1 comprises        the amino acid sequence DYWMY (SEQ ID NO: 164); CDR2 comprises        the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233) and        CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO: 300).    -   A Nanobody in accordance with XXXVIII), in which        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).    -   A Nanobody in accordance with XXXVIII), which is a GLEW-class        Nanobody.    -   A Nanobody in accordance with XXXVIII), which contains an        arginine residue (R) at position 103.    -   A Nanobody in accordance with XXXVIII), which has at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with one of the amino acid sequences of SEQ ID NO's 52 (TNF1),        76 (TNF13), 77 (TNF14), 95 (TNF29) or 96 (TNF30).    -   A Nanobody in accordance with XXXVIII), which is a humanized        Nanobody.    -   A Nanobody in accordance with XXXVIII), which contains a leucine        residue (L) at position 108.    -   A Nanobody in accordance with XXXVIII), which has a K_(off) rate        for TNF of better than 2.10-3 (1/s), preferably better than        1.10-3 (1/s); or a humanized variant of such a Nanobody;    -   A Nanobody in accordance with XXXVIII), which has an EC50 value        in the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than the EC50 value of        Nanobody VHH 3E (SEQ ID NO:4) of WO 04/041862 in the same assay;        or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with XXXVIII), which has an EC50 value        in the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than 5 nM; or a        humanized variant of such a Nanobody.    -   A Nanobody in accordance with XXXVIII), which has an EC50 value        in the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than 3 nM; or a        humanized variant of such a Nanobody.-   XXXIX) A Nanobody against TNF-alpha, which consist of 4 framework    regions (FR1 to FR4 respectively) and 3 complementarity determining    regions (CDR1 to CDR3 respectively), in which    -   a) CDR1 is:        -   the amino acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence DYWMY (SEQ ID NO: 164); or        -   an amino acid sequences that has only 1 amino acid            difference with the amino acid sequence DYWMY (SEQ ID NO;            164);    -   and in which:    -   b) CDR2 is:        -   the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence EINTNGLITKYPDSVKG            (SEQ ID NO: 233);    -   and in which    -   c) CDR3 is:        -   the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SPSGFN (SEQ ID NO: 300); or        -   an amino acid sequences that has only 1 amino acid            difference with the amino acid sequence SPSGFN (SEQ ID NO:            300).    -   A Nanobody in accordance with XXXIX), in which CDR1 is the amino        acid sequence DYWMY (SEQ ID NO: 164).    -   A Nanobody in accordance with XXXIX), in which CDR2 is the amino        acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233).    -   A Nanobody in accordance with XXXIX), in which CDR3 is the amino        acid sequence SPSGFN (SEQ ID NO: 300)    -   A Nanobody in accordance with XXXIX), in which:        -   CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); and            CDR3 is the amino acid sequence SPSGFN (SEQ ID NO: 300); or        -   CDR1 is the amino acid sequence DYWMY (SEQ ID NO: 164); and            CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID            NO: 233); or        -   CDR2 is the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID            NO: 233); and CDR3 is the amino acid sequence SPSGFN (SEQ ID            NO: 300)    -   A Nanobody in accordance with XXXIX), in which CDR1 is the amino        acid sequence DYWMY (SEQ ID NO: 164); and CDR3 is the amino acid        sequence SPSGFN (SEQ ID NO: 300).    -   A Nanobody in accordance with XXXIX), in which CDR1 is the amino        acid sequence DYWMY (SEQ ID NO: 164); CDR2 is the amino acid        sequence EINTNGLITKYPDSVKG (SEQ ID NO: 233) and CDR3 is the        amino acid sequence SPSGFN (SEQ ID NO: 300).    -   A Nanobody in accordance with XXXIX), in which:        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).    -   A Nanobody in accordance with XXXIX), which is a GLEW-class        Nanobody.    -   A Nanobody in accordance with XXXIX), which contains an arginine        residue (R) at position 103.    -   A Nanobody in accordance with XXXIX), which has at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with one of the amino acid sequences of SEQ ID NO's 52 (TNF1),        76 (TNF13), 77 (TNF14), 95 (TNF29) or 96 (TNF30).    -   A Nanobody in accordance with XXXIX), which is a humanized        Nanobody.    -   A Nanobody in accordance with XXXIX), which contains a leucine        residue (L) at position 108.    -   A Nanobody in accordance with XXXIX), which has a K_(off) rate        for TNF of better than 2.10-3 (1/s), preferably better than        1.10-3 (1/s); or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with XXXIX), which has an EC50 value in        the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than the EC50 value of        Nanobody VHH 3E (SEQ ID NO:4) of WO 04/041862 in the same assay        or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with XXXIX), which has an EC50 value in        the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than 5 nM; or a        humanized variant of such a Nanobody.    -   A Nanobody in accordance with XXXIX), which has an EC50 value in        the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than 3 nM; or a        humanized variant of such a Nanobody.    -   A Nanobody in accordance with XXXIX), which is chosen from the        group consisting of TNF 13 (SEQ ID NO: 76), TNF 14 (SEQ ID NO:        77), TNF 29 (SEQ ID NO: 95) and TNF 30 (SEQ ID NO:96).    -   A Nanobody in accordance with XXXIX), which is TNF 30 (SEQ ID        NO: 96);        with some other preferred aspects being:-   XL) A protein or polypeptide, which comprises or essentially    consists of a Nanobody in accordance with XXXVIII) or XXXIX).-   XLI) A protein or polypeptide, which comprises or essentially    consists of at least one Nanobody in accordance with XXXVIII) or    XXXIX).    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX).    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX), and which is such that said protein or polypeptide,        upon binding to a TNF trimer, is capable inhibiting or reducing        the TNF receptor crosslinking that is mediated by said TNF        trimer and/or the signal transduction that is mediated by such        receptor crosslinking.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX), and which is capable of intramolecular binding to at        least two TNF receptor binding sites on a TNF trimer.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX), which are directly linked to each other or linked to        each other via a linker.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX), which are linked to each other via an amino acid        sequence.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX), which are linked to each other via an amino acid        sequence (such as, without limitation, an amino acid sequence        that comprises glycine and serine residues) that comprises at        least 14 amino acids, more preferably at least 17 amino acids,        such as about 20-40 amino acids (such as the linker GS30).    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises or essentially consists of the polypeptide        TNF 7 (SEQ ID NO: 73), in which both Nanobodies TNF 1 have been        humanized.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises or essentially consists the polypeptide        TNF 55 (SEQ ID NO: 419) or TNF 56 (SEQ ID NO: 420).    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which is pegylated.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX), and which is such that said protein or polypeptide,        upon binding to a TNF trimer, is capable inhibiting or reducing        the TNF receptor crosslinking that is mediated by said TNF        trimer and/or the signal transduction that is mediated by such        receptor crosslinking; and/or which is such that said protein or        polypeptide is capable of intramolecular binding to at least two        TNF receptor binding sites on a TNF trimer, and which protein or        polypeptide further comprises at least one Nanobody directed        against human serum albumin.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX), and which protein or polypeptide further comprises at        least one Nanobody directed against human serum albumin, in        which the two Nanobodies in accordance with XXXVIII) or XXXIX)        are linked to each other via the at least one Nanobody directed        against human serum albumin, and in which the two Nanobodies in        accordance with XXXVIII) or XXXIX) are either linked directly to        the at least one Nanobody directed against human serum albumin,        or are linked to the at least one Nanobody directed against        human serum albumin via a linker.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX), and which protein or polypeptide further comprises at        least one Nanobody directed against human serum albumin, in        which the two Nanobodies in accordance with XXXVIII) or XXXIX)        are linked to each other via the at least one Nanobody directed        against human serum albumin, and in which the two Nanobodies in        accordance with XXXVIII) or XXXIX) are either linked directly to        the at least one Nanobody directed against human serum albumin,        or are linked to the at least one Nanobody directed against        human serum albumin via a linker, in which the linker is an        amino acid sequence (such as, without limitation, a linker that        comprises glycine and serine residues), and in particular an        amino acid sequence that comprises between 3 and 40 amino acid        residues, such as between 5 and 15 amino acid residues (such as        the linker GS9).    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises two Nanobodies in accordance with XXXVIII)        or XXXIX), and which protein or polypeptide further comprises at        least one Nanobody directed against human serum albumin, in        which the two Nanobodies in accordance with XXXVIII) or XXXIX)        are linked to each other via the at least one Nanobody directed        against human serum albumin, and in which the two Nanobodies in        accordance with XXXVIII) or XXXIX) are either linked directly to        the at least one Nanobody directed against human serum albumin,        or are linked to the at least one Nanobody directed against        human serum albumin via a linker, and which protein or        polypeptide is such that said protein or polypeptide, upon        binding to a TNF trimer, is capable inhibiting or reducing the        TNF receptor crosslinking that is mediated by said TNF trimer        and/or the signal transduction that is mediated by such receptor        crosslinking; and/or which protein or polypeptide is such that        said protein or polypeptide is capable of intramolecular binding        to at least two TNF receptor binding sites on a TNF trimer.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), in which the at least one Nanobody directed against human        serum albumin is a humanized Nanobody.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), in which the at least one Nanobody directed against human        serum albumin is a humanized variant of the Nanobody ALB 1 (SEQ        ID NO: 63).    -   A protein or polypeptide in accordance with any one of XL) or        XLI), in which the at least one Nanobody directed against human        serum albumin is a chosen from the group consisting of ALB 3        (SEQ ID NO: 87), ALB 4 (SEQ ID NO: 88), ALB 5 (SEQ ID NO: 89),        ALB 6 (SEQ ID NO: 100), ALB 7 (SEQ ID NO: 101), ALB 8 (SEQ ID        NO: 102) ALB 9 (SEQ ID NO: 103) and ALB 10 (SEQ ID NO: 104).    -   A protein or polypeptide in accordance with any one of XL) or        XLI), in which the at least one Nanobody directed against human        serum albumin is ALB 8.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises or essentially consists of two humanized        Nanobodies in accordance with any one of XL) or XLI) and one        humanized variant of the Nanobody ALB 1 (SEQ ID NO: 63).    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises or essentially consists of the polypeptide        TNF24 (SEQ ID NO: 90), in which both the Nanobody TNF 1 as well        as the Nanobody ALB 1 has been humanized.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises or essentially consists of two Nanobodies        TNF 30 and one Nanobody ALB 8.    -   A protein or polypeptide in accordance with any one of XL) or        XLI), which comprises or essentially consists the polypeptide        TNF 60 (SEQ ID NO: 417).

It should be noted that when a Nanobody is mentioned above as being “inaccordance with XXXVIII” or “in accordance with XXXIX”, it is at leastaccording to one of XXXVIII) and/or XXXIX), and may also include any oneor more of the other aspects that are indicated as being “in accordancewith XXXVIII” or “in accordance with XXXIX” above. Similarly, when aprotein or polypeptide is mentioned above as being “in accordance withany one of XL) or XLI)”, it is at least according to one of XL) to XLI),may be according to two or more of VI) to XVIII), and may also includeany one or more of the other aspects that are indicated as being “inaccordance with any one of XL) or XLI) above.

For the Nanobodies based on Nanobody TNF1 above (including but notlimited to the humanized Nanobodies), the framework sequences maygenerally be as described herein, and preferably are as follows:

a) FR1 comprises or is:

-   -   the amino acid sequence of SEQ ID NO: 130; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence of SEQ        ID NO: 130; or    -   an amino acid sequences that has only 1 amino acid difference        with the amino acid sequence of SEQ ID NO: 130;

and

b) FR2 comprises or is:

-   -   the amino acid sequence of SEQ ID NO: 198; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence of SEQ        ID NO: 198; or    -   an amino acid sequences that has 2 or only 1 amino acid        difference(s) with the amino acid sequence of SEQ ID NO: 198;

and

c) FR3 comprises or is:

-   -   the amino acid sequence of SEQ ID NO: 266; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence of SEQ        ID NO: 266; or    -   an amino acid sequences that has only 1 amino acid difference        with the amino acid sequence of SEQ ID NO: 266.

and

d) FR4 comprises or is:

-   -   the amino acid sequence of SEQ ID NO: 334; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence of SEQ        ID NO: 334; or    -   an amino acid sequences that has only 1 amino acid difference        with the amino acid sequence of SEQ ID NO: 334;

in which the amino acid differences present in the framework sequencesare more preferably as described herein.

Nanobodies against TNF-alpha, which have framework regions as describedabove (i.e. similar to TNF1), and in which at least one of the frameworkregions (such as any two, any three or all four framework regions) havebeen humanized, form a further aspect of the invention. Such Nanobodiesmay in particular have CDR's that are such that the Nanobody has a Koffrate for TNF of better than 2.10-3 (1/s), preferably better than 1.10-3(1/s); and/or have CDR's that are such that the Nanobody has an EC50value in the cell-based assay using KYM cells described in Example 1,under 3), of WO 04/041862 that is better than the EC50 value of NanobodyVHH 3E (SEQ ID NO:4) of WO 04/041862 in the same assay; and inparticular better than 12 nM, more in particular better than 5 nM, evenmore in particular better than 3 nM. Also, or alternatively, suchNanobodies are preferably directed against the same epitope of TNF (i.e.the TNF trimer) as TNF1.

In particular, the invention relates to a Nanobody against TNF-alpha,which is a humanized variant of a Nanobody against TNF-alpha, whichNanobody against TNF-alpha has the following framework sequences: FR1:SEQ ID NO: 130; FR2: SEQ ID NO: 198; FR3: SEQ ID NO: 266; and FR4: SEQID NO: 334. Such a Nanobody may in particular have CDR's that are suchthat the Nanobody has a Koff rate for TNF of better than 2.10-3 (1/s),preferably better than 1.10-3 (1/s); and/or have CDR's that are suchthat the Nanobody has an EC50 value in the cell-based assay using KYMcells described in Example 1, under 3), of WO 04/041862 that is betterthan the EC50 value of Nanobody VHH 3E (SEQ ID NO:4) of WO 04/041862 inthe same assay; and in particular better than 12 nM, more in particularbetter than 5 nM, even more in particular better than 3 nM. Also, oralternatively, such Nanobodies are preferably directed against the sameepitope of TNF (i.e. the TNF trimer) as TNF1.

Another clone that has been found to be particularly useful as ananti-TNF Nanobody is the clone PMP5F10 (TNF3, SEQ ID NO: 60). As can beseen from the comparative data from the KYM-assay in Table 39, TNF3 hasan EC50 value that is more than 15 times better than the best monovalentNanobody described in WO 04/41862.

Accordingly, Nanobodies that comprise one or more, preferably any twoand more preferably all three of the CDR's present in the clone PMP5F10(or CDR sequences that are derived therefrom or correspond thereto) areparticularly preferred in the invention. Also, these Nanobodiespreferably belong to the KERE class.

More specifically, some preferred aspects of this embodiment of theinvention are:

-   XLII) A Nanobody against TNF-alpha, which consist of 4 framework    regions (FR1 to FR4 respectively) and 3 complementarity determining    regions (CDR1 to CDR3 respectively), in which    -   a) CDR1 comprises:        -   the amino acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence NYYMG (SEQ ID NO:            172);    -   and    -   b) CDR2 comprises:        -   the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence NISWRGYNIYYKDSVKG            (SEQ ID NO: 240);    -   and    -   c) CDR3 comprises:        -   the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SILPLSDDPGWNTY (SEQ            ID NO: 308).    -   A Nanobody in accordance with XLII), in which CDR1 comprises the        amino acid sequence NYYMG (SEQ ID NO: 172).    -   A Nanobody in accordance with XLII), in which CDR2 comprises the        amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240).    -   A Nanobody in accordance with XLII), in which CDR3 comprises the        amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308).    -   A Nanobody in accordance with XLII), in which:        -   CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO:            172); and CDR3 comprises the amino acid sequence            SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   CDR1 comprises the amino acid sequence NYYMG (SEQ ID NO:            172); and CDR2 comprises the amino acid sequence            NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or        -   CDR2 comprises the amino acid sequence NISWRGYNIYYKDSVKG            (SEQ ID NO: 240); and CDR3 comprises the amino acid sequence            SILPLSDDPGWNTY (SEQ ID NO: 308).    -   A Nanobody in accordance with XLII), in which CDR1 comprises the        amino acid sequence NYYMG (SEQ ID NO: 172); CDR2 comprises the        amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308) and CDR3        comprises the amino acid sequence ILPLSDDPGWNTY (SEQ ID NO 436).    -   A Nanobody in accordance with XLII), in which        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).    -   A Nanobody in accordance with XLII), which is a KERE-class        Nanobody.    -   A Nanobody in accordance with XLII), which has at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with one of the amino acid sequences of SEQ ID NO's 50 (TNF3),        83 (TNF20), 85 (TNF21), 85 (TNF22), 96 (TNF23) or 99 (TNF33).    -   A Nanobody in accordance with XLII), which is a humanized        Nanobody.    -   A Nanobody in accordance with XLII), which has a K_(off) rate        for TNF of better than 2.10⁻³ (1/s), preferably better than        1.10⁻³ (1/s); or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with XLII), which has an EC50 value in        the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than the EC50 value of        Nanobody VHH 3E (SEQ ID NO:4) of WO 04/041862 in the same assay;        or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with XLII), which has an EC50 value in        the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than 5 nM; or a        humanized variant of such a Nanobody.    -   A Nanobody in accordance with XLII), which has an EC50 value in        the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than 3 nM; or a        humanized variant of such a Nanobody.-   XLIII) A Nanobody against TNF-alpha, which consist of 4 framework    regions (FR1 to FR4 respectively) and 3 complementarity determining    regions (CDR1 to CDR3 respectively), in which    -   a) CDR1 is:        -   the amino acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NYYMG (SEQ ID NO: 172); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence NYYMG (SEQ ID NO:            172);    -   and    -   b) CDR2 is:        -   the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240);            or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference(s) with the amino acid sequence NISWRGYNIYYKDSVKG            (SEQ ID NO: 240);    -   and    -   c) CDR3 is:        -   the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequence that has at least 80%, preferably at            least 90%, more preferably at least 95%, even more            preferably at least 99% sequence identity with the amino            acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308); or        -   an amino acid sequences that has 2 or only 1 amino acid            difference with the amino acid sequence SILPLSDDPGWNTY (SEQ            ID NO: 308).    -   A Nanobody in accordance with XLIII), in which CDR1 is the amino        acid sequence NYYMG (SEQ ID NO: 172).    -   A Nanobody in accordance with XLIII), in which CDR2 is the amino        acid sequence NISWRGYNIYYKDSVKG (SEQ ID NO: 240).    -   A Nanobody in accordance with XLIII), in which CDR3 is the amino        acid sequence SILPLSDDPGWNTY (SEQ ID NO: 308).    -   A Nanobody in accordance with XLIII), in which:        -   CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); and            CDR3 is the amino acid sequence SILPLSDDPGWNTY (SEQ ID NO:            308); or        -   CDR1 is the amino acid sequence NYYMG (SEQ ID NO: 172); and            CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID            NO: 240); or        -   CDR2 is the amino acid sequence NISWRGYNIYYKDSVKG (SEQ ID            NO: 240); and CDR3 is the amino acid sequence SILPLSDDPGWNTY            (SEQ ID NO: 308).    -   A Nanobody in accordance with XLIII), in which CDR1 is the amino        acid sequence NYYMG (SEQ ID NO: 172); CDR2 is the amino acid        sequence SILPLSDDPGWNTY (SEQ ID NO: 308) and CDR3 is the amino        acid sequence ILPLSDDPGWNTY (SEQ ID NO: 436).    -   A Nanobody in accordance with XLIII), in which        -   any amino acid substitution is preferably a conservative            amino acid substitution; and/or        -   said amino acid sequence preferably only contains amino acid            substitutions, and no amino acid deletions or insertions,            compared to the above amino acid sequence(s).    -   A Nanobody in accordance with XLIII), which is a KERE-class        Nanobody.    -   A Nanobody in accordance with XLIII), which has at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with one of the amino acid sequences of SEQ ID NO's 50 (TNF3),        83 (TNF20), 85 (TNF21), 85 (TNF22), 96 (TNF23) or 99 (TNF33).    -   A Nanobody in accordance with XLIII), which is a humanized        Nanobody.    -   A Nanobody in accordance with XLIII), which has a K_(off) rate        for TNF of better than 2.10⁻³ (1/s), preferably better than        2.10⁻³ (1/s); or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with XLIII), which has an EC50 value in        the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than the EC50 value of        Nanobody VHH 3E (SEQ ID NO:4) of WO 04/041862 in the same assay;        or a humanized variant of such a Nanobody.    -   A Nanobody in accordance with XLIII), which has an EC50 value in        the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than 5 nM; or a        humanized variant of such a Nanobody.    -   A Nanobody in accordance with XLIII), which has an EC50 value in        the cell-based assay using KYM cells described in Example 1,        under 3), of WO 04/041862 that is better than 3 nM; or a        humanized variant of such a Nanobody.    -   A Nanobody in accordance with XLIII), which is chosen from the        group consisting of SEQ ID NO's, 83 (TNF20), 85 (TNF21), 85        (TNF22), 96 (TNF23) or 98 (TNF33)TNF 13 (SEQ ID NO: 76), TNF 14        (SEQ ID NO: 77), TNF 29 (SEQ ID NO: 95) and TNF 30 (SEQ ID        NO:96)        with some other preferred aspects being:-   XLIV) A protein or polypeptide, which comprises or essentially    consists of a Nanobody in accordance with XLII) or XLIII).-   XLV) A protein or polypeptide, which comprises or essentially    consists of at least one Nanobody in accordance with XLII) or    XLIII).-   XLVI) A protein or polypeptide, which comprises two Nanobodies in    accordance with XLII) or XLIII).-   XLVII) A protein or polypeptide, which comprises two Nanobodies in    accordance with XLII) or XLIII), and which is such that said protein    or polypeptide, upon binding to a TNF trimer, is capable inhibiting    or reducing the TNF receptor crosslinking that is mediated by said    TNF trimer and/or the signal transduction that is mediated by such    receptor crosslinking.-   XLVIII) A protein or polypeptide, which comprises two Nanobodies in    accordance with XLII) or XLIII), and which is capable of    intramolecular binding to at least two TNF receptor binding sites on    a TNF trimer.    -   A protein or polypeptide in accordance with any one of XLIV) or        XLVIII), which comprises or essentially consists of the        polypeptide TNF 6 (SEQ ID NO: 72) or TNF 9 (SEQ ID NO: 75, in        which both Nanobodies TNF 3 have been humanized    -   A protein or polypeptide in accordance with any one of XLIV) or        XLVIII), which is pegylated.    -   A protein or polypeptide, which comprises two Nanobodies in        accordance with XLII) or XLIII), and which is such that said        protein or polypeptide, upon binding to a TNF trimer, is capable        inhibiting or reducing the TNF receptor crosslinking that is        mediated by said TNF trimer and/or the signal transduction that        is mediated by such receptor crosslinking; and/or which is such        that said protein or polypeptide is capable of intramolecular        binding to at least two TNF receptor binding sites on a TNF        trimer, and which protein or polypeptide further comprises at        least one Nanobody directed against human serum albumin.    -   A protein or polypeptide in accordance with any one of XLIV) or        XLVIII), in which the at least one Nanobody directed against        human serum albumin is a humanized Nanobody.    -   A protein or polypeptide in accordance with any one of XLIV) or        XLVIII), in which the at least one Nanobody directed against        human serum albumin is a humanized variant of the Nanobody ALB 1        (SEQ ID NO: 63).    -   A protein or polypeptide in accordance with any one of XLIV) or        XLVIII), in which the at least one Nanobody directed against        human serum albumin is a chosen from the group consisting of ALB        3 (SEQ ID NO: 87), ALB 4 (SEQ ID NO: 88), ALB 5 (SEQ ID NO: 89),        ALB 6 (SEQ ID NO: 100), ALB 7 (SEQ ID NO: 101), ALB 8 (SEQ ID        NO: 102) ALB 9 (SEQ ID NO: 103) and ALB 10 (SEQ ID NO: 104).    -   A protein or polypeptide in accordance with any one of XLIV) or        XLVIII), in which the at least one Nanobody directed against        human serum albumin is ALB 8.    -   A protein or polypeptide in accordance with any one of XLIV) or        XLVIII), which comprises or essentially consists of two        humanized Nanobodies in accordance with any one of XLIV)        or XLVIII) and one humanized variant of the Nanobody ALB 1 (SEQ        ID NO: 63).    -   A protein or polypeptide in accordance with any one of XLIV) or        XLVIII), which comprises or essentially consists of the        polypeptide TNF26 (SEQ ID NO: 92), in which both the Nanobodies        TNF 3 as well as the Nanobody ALB 1 has been humanized.

It should be noted that when a Nanobody is mentioned above as being “inaccordance with XLII” or “in accordance with XLIII”, it is at leastaccording to one of XLII) and/or XLIII), and may also include any one ormore of the other aspects that are indicated as being “in accordancewith XLII)” or “in accordance with XLIII)” above. Similarly, when aprotein or polypeptide is mentioned above as being “in accordance withany one of XLIV) or XLVIII)”, it is at least according to one of XL) toXLI), may be according to two or more of XLIV) to XLVIII), and may alsoinclude any one or more of the other aspects that are indicated as being“in accordance with any one of XLIV) or XLVIII) above.

For the Nanobodies based on Nanobody TNF3 above (including but notlimited to the humanized Nanobodies), the framework sequences maygenerally be as described herein, and preferably are as follows:

a) FR1 comprises or is:

-   -   the amino acid sequence of SEQ ID NO: 138; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence of SEQ        ID NO: 138; or    -   an amino acid sequences that has only 1 amino acid difference        with the amino acid sequence of SEQ ID NO: 138;

and

b) FR2 comprises or is:

-   -   the amino acid sequence of SEQ ID NO: 206; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence of SEQ        ID NO: 206; or    -   an amino acid sequences that has 2 or only 1 amino acid        difference(s) with the amino acid sequence of SEQ ID NO: 206;

and

c) FR3 comprises or is:

-   -   the amino acid sequence of SEQ ID NO: 274; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence of SEQ        ID NO: 274; or    -   an amino acid sequences that has only 1 amino acid difference        with the amino acid sequence of SEQ ID NO: 274.

and

d) FR4 comprises or is:

-   -   the amino acid sequence of SEQ ID NO: 342; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence of SEQ        ID NO: 342; or    -   an amino acid sequences that has only 1 amino acid difference        with the amino acid sequence of SEQ ID NO: 342;        in which the amino acid differences present in the framework        sequences are more preferably as described herein.

In another aspect, the invention relates to a Nanobody with an aminoacid sequence that is chosen from the group consisting of SEQ ID NO's:52 to 60, from the group consisting of SEQ ID NO's: 76 to 86, from thegroup consisting of SEQ ID NO's: 95 to 99, from the group consisting ofSEQ ID NO's 105 to 129 or from the group consisting of from amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one or more of the amino acid sequences of SEQ IDNO's: 52 to 60, SEQ ID NO's: 76 to 86, SEQ ID NO's: 95 to 99 or SEQ IDNO's 105 to 129, in which the latter amino acid sequences mostpreferably have framework sequences that are as further defined belowunder the general description of the framework sequences of Nanobodies.

According to a specific, but non-limiting embodiment, the latter aminoacid sequences are “humanized”, as further described herein.

Most preferably, the Nanobodies of the invention are chosen from thegroup consisting of SEQ ID NO's: 52 to 60, from the group consisting ofSEQ ID NO's: 76 to 86, from the group consisting of SEQ ID NO's: 95 to99, or from the group consisting of SEQ ID NO's 105 to 129, of which the“humanized” Nanobodies of SEQ ID NO's 76 to 86 and SEQ ID NO's: 95 to 99may be particularly preferred.

As mentioned above, a particularly preferred Nanobody of the inventionis the clone PMP1C2 (TNF1; SEQ ID NO: 52). Thus, in a preferred aspect,the invention relates to a Nanobody with an amino acid sequence that ischosen from the group consisting of SEQ ID NO: 52 or from the groupconsisting of from amino acid sequences that have more than 80%,preferably more than 90%, more preferably more than 95%, such as 99% ormore “sequence identity” (as defined herein) with the amino acidsequence of SEQ ID NO:52, in which the latter amino acid sequences mostpreferably have framework sequences that are as further defined belowunder the general description of the framework sequences of Nanobodies.

Particularly preferred are humanized variants of the clone PMP1C2 (TNF1;SEQ ID NO: 52). Some preferred, but non-limiting examples of suchhumanized variants are the clones TNF13 (SEQ ID NO: 76), TNF14 (SEQ IDNO:77), TNF29 (SEQ ID NO: 95) and TNF 30 (SEQ ID NO: 96). Thus, in apreferred aspect, the invention relates to a humanized Nanobody with anamino acid sequence that is chosen from the group consisting of SEQ IDNO's: 76, 77, 95 or 96, or from the group consisting of from amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one of the amino acid sequences of SEQ ID NO's: 76,77, 95 or 96, in which the latter amino acid sequences most preferablyhave framework sequences that are as further defined below under thegeneral description of the framework sequences of Nanobodies.

According to one preferred embodiment, the Nanobody of the invention isa humanized variant of the Nanobody TNF 1 (SEQ ID NO: 52).

Some preferred aspects of this embodiment of the invention are:

-   XLIX) A humanized variant of the Nanobody TNF 1, which has a Koff    rate for TNF of better than 2.10-3 (1/s), preferably better than    1.10-3 (1/s).-   L) A humanized variant of the Nanobody TNF 1, which has an EC50    value in the cell-based assay using KYM cells described in Example    1, under 3), of WO 04/041862 that is better than the EC50 value of    Nanobody VHH 3E (SEQ ID NO:4) of WO 04/041862 in the same assay.-   LI) A humanized variant of the Nanobody TNF 1, which has an EC50    value in the cell-based assay using KYM cells described in Example    1, under 3), of WO 04/041862 that is better than 5 nM.-   LII) A humanized variant of the Nanobody TNF 1, which has an EC50    value in the cell-based assay using KYM cells described in Example    1, under 3), of WO 04/041862 that is better than 3 nM.-   LIII) A protein or polypeptide, which comprises or essentially    consists of at least one Nanobody which is a humanized variant of    the Nanobody TNF 1 in accordance with any one of XLIX) to LII)-   LIV) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 1 in accordance with any one of XLIX) to LII)    (optionally linked via a linker).-   LV) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 1 in accordance with any one of XLIX) to LII)    (optionally linked via a linker), and which is such that said    protein or polypeptide, upon binding to a TNF trimer, is capable    inhibiting or reducing the TNF receptor crosslinking that is    mediated by said TNF trimer and/or the signal transduction that is    mediated by such receptor crosslinking.-   LVI) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 1 in accordance with any one of XLIX) to LII) and which    is capable of intramolecular binding to at least two TNF receptor    binding sites on a TNF trimer.-   LVII) A protein or polypeptide which comprises or essentially    consists of the polypeptide TNF 7 (SEQ ID NO: 73), in which both    Nanobodies TNF 1 have been humanized.-   LVIII) A protein or polypeptide which comprises or essentially    consists of the polypeptide TNF 7 (SEQ ID NO: 73), in which both    Nanobodies TNF 1 have been humanized, and which is pegylated.-   LIX) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 1 in accordance with any one of XLIX) to LII), and    which further comprises at least one Nanobody directed against human    serum albumin.-   LX) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 1 in accordance with any one of XLIX) to LII), which    are each linked (optionally linked via a linker) to one Nanobody    directed against human serum albumin.    -   A protein or polypeptide in accordance with any one of LIII) to        LX), in which the at least one Nanobody directed against human        serum albumin is a humanized Nanobody.    -   A protein or polypeptide in accordance with any one of LIII) to        LX), in which the at least one Nanobody directed against human        serum albumin is a humanized variant of the Nanobody ALB 1 (SEQ        ID NO: 63).    -   A protein or polypeptide in accordance with any one of LIII) to        LX), in which the at least one Nanobody directed against human        serum albumin is a chosen from the group consisting of ALB 3        (SEQ ID NO: 87), ALB 4 (SEQ ID NO: 88), ALB 5 (SEQ ID NO: 89),        ALB 6 (SEQ ID NO: 100), ALB 7 (SEQ ID NO: 101), ALB 8 (SEQ ID        NO: 102) ALB 9 (SEQ ID NO: 103) and ALB 10 (SEQ ID NO: 104).    -   A protein or polypeptide in accordance with any one of LIII) to        LX), in which the at least one Nanobody directed against human        serum albumin is ALB 8.    -   A protein or polypeptide in accordance with any one of LIII) to        LX), which comprises or essentially consists of the polypeptide        TNF24 (SEQ ID NO: 90), in which both the Nanobody TNF 1 as well        as the Nanobody ALB TNF 1 has been humanized.    -   A protein or polypeptide in accordance with any one of LIII) to        LX), which comprises or essentially consists of two Nanobodies        TNF 30 and one Nanobody ALB 8.

According to one preferred embodiment, the Nanobody of the invention isa humanized variant of the Nanobody TNF 3 (SEQ ID NO: 60).

Some preferred aspects of this embodiment of the invention are:

-   LXI) A humanized variant of the Nanobody TNF 3, which has a Koff    rate for TNF of better than 2.10-3 (1/s), preferably better than    1.10-3 (1/s).-   LXII) A humanized variant of the Nanobody TNF 3, which has an EC50    value in the cell-based assay using KYM cells described in Example    1, under 3), of WO 04/041862 that is better than the EC50 value of    Nanobody VHH 3E (SEQ ID NO:4) of WO 04/041862 in the same assay.-   LXIII) A humanized variant of the Nanobody TNF 3, which has an EC50    value in the cell-based assay using KYM cells described in Example    1, under 3), of WO 04/041862 that is better than 5 nM.-   LXIV) A humanized variant of the Nanobody TNF 3, which has an EC50    value in the cell-based assay using KYM cells described in Example    1, under 3), of WO 04/041862 that is better than 3 nM.-   LXV) A protein or polypeptide, which comprises or essentially    consists of at least one Nanobody which is a humanized variant of    the Nanobody TNF 3 in accordance with any one of LXI) to LXIV)-   LXVI) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 3 in accordance with any one of LXI) to LXIV)    (optionally linked via a linker).-   LXVII) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 3 in accordance with any one of LXI) to LXIV)    (optionally linked via a linker), and which is such that said    protein or polypeptide, upon binding to a TNF trimer, is capable    inhibiting or reducing the TNF receptor crosslinking that is    mediated by said TNF trimer and/or the signal transduction that is    mediated by such receptor crosslinking.-   LXVIII) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 3 in accordance with any one of LXI) to LXIV) and which    is capable of intramolecular binding to at least two TNF receptor    binding sites on a TNF trimer.-   LXIX) A protein or polypeptide which comprises or essentially    consists of the polypeptide TNF 6 (SEQ ID NO: 72) or TNF 9 (SEQ ID    NO: 75), in which both Nanobodies TNF 3 have been humanized.-   LXX) A protein or polypeptide which comprises or essentially    consists of the polypeptide TNF 6 (SEQ ID NO: 72) or TNF 9 (SEQ ID    NO: 75), in which both Nanobodies TNF 3 have been humanized, and    which is pegylated.-   LXXI) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 3 in accordance with any one of LXI) to LXIV), and    which further comprises at least one Nanobody directed against human    serum albumin.-   LXXII) A protein or polypeptide, which comprises or essentially    consists of two Nanobodies which are humanized variants of the    Nanobody TNF 3 in accordance with any one of LXI) to LXIV), which    are each linked (optionally linked via a linker) to one Nanobody    directed against human serum albumin.    -   A protein or polypeptide in accordance with any one of LXV) to        LXXII), in which the at least one Nanobody directed against        human serum albumin is a humanized Nanobody.    -   A protein or polypeptide in accordance with any one of LXV) to        LXXII), in which the at least one Nanobody directed against        human serum albumin is a humanized variant of the Nanobody ALB 1        (SEQ ID NO: 63).    -   A protein or polypeptide in accordance with any one of LXV) to        LXXII), in which the at least one Nanobody directed against        human serum albumin is a chosen from the group consisting of ALB        3 (SEQ ID NO: 87), ALB 4 (SEQ ID NO: 88), ALB 5 (SEQ ID NO: 89),        ALB 6 (SEQ ID NO: 100), ALB 7 (SEQ ID NO: 101), ALB 8 (SEQ ID        NO: 102) ALB 9 (SEQ ID NO: 103) and ALB 10 (SEQ ID NO: 104).    -   A protein or polypeptide in accordance with any one of LXV) to        LXXII), in which the at least one Nanobody directed against        human serum albumin is ALB 8.    -   A protein or polypeptide in accordance with any one of LXV) to        LXXII), which comprises or essentially consists of the        polypeptide TNF26 (SEQ ID NO: 92), in which both the Nanobodies        TNF 3 as well as the Nanobody ALB 1 has been humanized.

In another aspect, the invention relates to a polypeptide that comprisesor essentially consists of at least one Nanobody against TNF-alpha asdefined herein. Such polypeptides are also referred to herein as“polypeptides of the invention” and may be as further describedhereinbelow and/or as generally described in WO 04/041862 for theNanobodies disclosed therein, and may for example be multivalentpolypeptides or multispecific polypeptides, again as further describedhereinbelow.

Preferably, a polypeptide of the invention is either bivalent ortrivalent (i.e. comprising two or three Nanobodies of the invention,respectively, optionally linked via one or two linkers as defined below,respectively) or a multispecific polypeptide, comprising one or two, andpreferably two, Nanobodies of the invention and at least one Nanobodydirected against a serum protein, and in particular against a humanserum protein, such as against human serum albumin.

In one preferred, but non-limiting embodiments, the Nanobodies of theinvention present in the polypeptides of the invention are chosen fromthe group consisting of SEQ ID NO's: 52 to 60 and SEQ ID NO's 105-129 orfrom humanized variants thereof, and in particular from the “humanized”Nanobodies of SEQ ID NO's 76 to 86 and SEQ ID NO's: 95 to 99. TheNanobodies against human serum albumin present in the polypeptides ofthe invention are preferably as defined below, and are more preferablychosen from the group consisting of SEQ ID NO's: 61 to 67, SEQ ID NO's:87 to 89 and SEQ ID NO's: 100-104, and in particular from the“humanized” Nanobodies against human serum albumin of SEQ ID NO's 76 to86 and SEQ ID NO's 100-104.

With respect to the Nanobodies that are present in the polypeptides ofthe invention, it will be clear to the skilled person that theNanobodies that are mentioned herein as “preferred” (or as “morepreferred”, “even more preferred”, etc.) are also preferred (or morepreferred, or even more preferred, etc.) for use in the polypeptidesdescribed herein. Thus, polypeptides that comprise or essentiallyconsist of one or more “preferred” Nanobodies of the invention willgenerally be preferred, and polypeptides that comprise or essentiallyconsist of one or more “more preferred” Nanobodies of the invention ofthe invention will generally be more preferred, etc.

Thus, in the invention, polypeptides that comprise one or moreNanobodies that essentially consist of one of the preferred variants ofclone PMP1C2 (TNF1; SEQ ID NO: 52), in which said preferred variants areas defined herein, are particularly preferred. Even more preferred arepolypeptides that comprise one or more Nanobodies that essentiallyconsist of one of the humanized variants of clone PMP1C2 (TNF1; SEQ IDNO: 52), in which said humanized variants are as defined herein(examples being, without limitation, TNF13, TNF14, TNF29 and TNF30).TNF30 is a particularly preferred humanized “building block” for use inthe polypeptides of the invention.

Some preferred, but non-limiting examples of such proteins andpolypeptides are PMP1C2 itself, the humanized variants TNF13, TNF14,TNF29 and TNF30; the constructs of SEQ ID NO: 70 (TNF4), SEQ ID NO: 73(TNF7), SEQ ID NO: 90 (TNF24), SEQ ID NO: 93 (TNF27); and the constructsof SEQ ID NO: 417 (TNF60), SEQ ID NO: 419 (TNF55) and SEQ ID NO: 420(TNF56), in which the latter three constructs contain the humanizedvariant TNF 30 as a building block.

As mentioned herein, the Nanobodies and constructs described herein maybe pegylated, or contain one or more (additional) amino acid residuesthat allow for pegylation and/or facilitate pegylation. Two preferred,but non-limiting examples of such polypeptides are TNF55 and TNF56,which both contain an additional cysteine residue for easy attachment ofa PEG-group.

Some preferred, but non-limiting examples of polypeptides of theinvention are the bivalent polypeptides of the invention of SEQ ID NO's:70 to 75 and the multispecific polypeptides of the invention of SEQ IDNO's: 90 to 94 and SEQ ID NO's 417 to 420.

As can be seen from the data represented below, and in particular fromthe data given in the Comparative Example, the Nanobodies and/orpolypeptides of the invention have improved properties. In particular,the proteins and polypeptides of the invention may have an improvedaffinity for human TNF-alpha (expressed as the EC₅₀-value in the KYMassay described herein), compared to the commercially available anti-TNFbiologicals Enbrel™, Humira™ and Remicade™. Also, the Nanobodiesdescribed herein may have an improved affinity for TNF-alpha compared tobest performing Nanobody described in the International application WO04/041862. It can thus be expected that polypeptides of the inventioncomprising at least one of the Nanobodies of the invention will alsohave improved properties compared to polypeptides that comprise only theNanobodies against TNF-alpha described in WO 04/041862.

More in particular, a polypeptide as described herein that comprises twoor more (and preferably two) Nanobodies as herein (and optionally forexample a Nanobody against human serum albumin), has an an EC50 value inthe cell-based assay using KYM cells described in Example 1, under 3),of WO 04/041862 that is better than the EC50 value of Humira® enRemicade®, and preferably also better than Enbrel® in the same assay.

For example, such a protein or polypeptide preferably has an EC50 valuein the cell-based assay using KYM cells described in Example 1, under3), of WO 04/041862 that is better than 0.2 nM, preferably better than0.1 nM, such as better than 0.7 Nm and in particular better than 0.4 nM.

It has also been shown by applicants that Nanobodies against mouseTNF-alpha and polypeptides comprising Nanobodies against mouse TNF-alphashow a beneficial biological activity in the following disease models(data not shown):

-   -   The dextran sulfate sodium (“DSS”) model of colitis, using both        regular mice as well as IL-10 knock out mice, as described by        Okayasu et al. (Gastroenterol 1990, 98(3): 694)    -   The collagen induced arthritis (“CIA”) model of arthritis        (“RA”), as described by Courtenay et al. (Nature 1980,        283(5748): 666), using both regular mice as well as IL-10 knock        out mice;    -   The IL-10 knockout mice model of IBD, as for example described        by Rennick et al. (Clin Immunol Immunopathol 1995, 76 (3 Pt 2):        S174)    -   The Kollias model of RA as for example described by Keffer et        al. (EMBO J 1991, 10(13): 4025)    -   The 2,4,6-trinitrobenzenesulphonic acid (“TNBS”) model of IBD,        as described by Elson et al. (J Immunol 1996, 157(5): 2174)    -   The CIA model of RA, described by Koppieters et al (manuscript        in preparation);    -   The synovial-derived fibroblast model (described below); and    -   The murine air pouch model.

Preferably, the Nanobodies described herein are better than Nanobody 1Afrom WO 04/041862 in at least one of these models, and preferably in allof these models; and more preferably Nanobody 3E from WO 04/041862 in atleast one of these models, and preferably in all of these models. Also,the polypeptides described herein are preferably equivalent to or betterthan Humira® or Remicade® in at least one of these models, andpreferably in all of these models; and more preferably also equivalentto or better than Enbrel® in at least one of these models, andpreferably in all of these models.

These data confirm that Nanobodies against TNF-alpha and polypeptidescontaining the same, such as the Nanobodies and polypeptides describedin WO 04/041862 and in particular the Nanobodies and polypeptidesdescribed herein, should have therapeutic efficacy against TNF mediateddiseases and disorders, such as the diseases and disorders mentionedabove.

In another aspect, the invention relates to a nucleic acid that encodesa Nanobody of the invention and/or a polypeptide of the invention. Sucha nucleic acid will also be referred to below as a “nucleic acid of theinvention” and may for example be in the form of a genetic construct, asdefined below.

In another aspect, the invention relates to host or host cell thatexpresses or is capable of expressing a Nanobody of the invention and/ora polypeptide of the invention; and/or that contains a nucleic acidencoding a Nanobody of the invention and/or a polypeptide of theinvention. Such a host or a host cell may also be analogous to the hostsand host cells described in WO 04/041862, but expressing or capable ofexpressing a Nanobody of the invention and/or a polypeptide of theinvention and/or containing a nucleic acid as described herein.

The invention further relates to a product or composition containing orcomprising a Nanobody of the invention, a polypeptide of the invention;and/or a nucleic acid of the invention. Such a product or compositionmay for example be a pharmaceutical composition (as described below) ora product or composition for diagnostic use (as also described below).Such a product or composition may also be analogous to the products andcompositions described in WO 04/041862, but containing or comprising aNanobody of the invention, a polypeptide of the invention or a nucleicacid of the invention.

The invention further relates to methods for preparing or generating theNanobodies, polypeptides, nucleic acids, host cells, products andcompositions as described herein, which methods are as further describedbelow. Also, generally, the Nanobodies, polypeptides, nucleic acids,host cells, products and compositions described herein may also beprepared and used in a manner analogous to the manner described in WO04/041862.

The invention further relates to applications and uses of the aboveNanobodies, polypeptides, nucleic acids, host cells, products andcompositions described herein, which applications and uses include, butare not limited to, the applications and uses described hereinbelowand/or the further uses and applications for Nanobodies againstTNF-alpha and/or for polypeptides containing the same in WO 04/041862.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description hereinbelow.

DETAILED DESCRIPTION OF THE INVENTION

The above and other aspects and embodiments of the invention will becomeclear from the further description hereinbelow, in which:

a) Unless indicated or defined otherwise, all terms used have theirusual meaning in the art, which will be clear to the skilled person.Reference is for example made to the standard handbooks, such asSambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd. Ed.),Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al,eds., “Current protocols in molecular biology”, Green Publishing andWiley Interscience, New York (1987); Lewin, “Genes II”, John Wiley &Sons, New York, N.Y., (1985); Old et al., “Principles of GeneManipulation: An Introduction to Genetic Engineering”, 2nd edition,University of California Press, Berkeley, Calif. (1981); Roitt et al.,“Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al.,Roitt's Essential Immunology, 10th Ed. Blackwell Publishing, U K (2001);and Janeway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, New York (2005), as well as to thegeneral background art cited herein;b) Unless indicated otherwise, the term “immunoglobulinsequence”—whether it used herein to refer to a heavy chain antibody orto a conventional 4-chain antibody—is used as a general term to includeboth the full-size antibody, the individual chains thereof, as well asall parts, domains or fragments thereof (including but not limited toantigen-binding domains or fragments such as V_(HH) domains orV_(H)/V_(L) domains, respectively). In addition, the term “sequence” asused herein (for example in terms like “immunoglobulin sequence”,“antibody sequence”, “variable domain sequence”, “V_(HH) sequence” or“protein sequence”), should generally be understood to include both therelevant amino acid sequence as well as nucleic acid sequences ornucleotide sequences encoding the same, unless the context requires amore limited interpretation;c) Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks, to the general background art referred to above andto the further references cited therein;d) Amino acid residues will be indicated according to the standardthree-letter or one-letter amino acid code, as mentioned in Table 1;

TABLE 1 one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0)Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0)Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimesalso considered to be a polar uncharged amino acid. ⁽²⁾Sometimes alsoconsidered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person.⁽⁴⁾As is known in the art, the charge of a His residue is greatlydependant upon even small shifts in pH, but a His residu can generallybe considered essentially uncharged at a pH of about 6.5.e) For the purposes of comparing two or more nucleotide sequences, thepercentage of “sequence identity” between a first nucleotide sequenceand a second nucleotide sequence may be calculated by dividing [thenumber of nucleotides in the first nucleotide sequence that areidentical to the nucleotides at the corresponding positions in thesecond nucleotide sequence] by [the total number of nucleotides in thefirst nucleotide sequence] and multiplying by [100%], in which eachdeletion, insertion, substitution or addition of a nucleotide in thesecond nucleotide sequence—compared to the first nucleotide sequence—isconsidered as a difference at a single nucleotide (position).

Alternatively, the degree of sequence identity between two or morenucleotide sequences may be calculated using a known computer algorithmfor sequence alignment such as NCBI Blast v2.0, using standard settings.

Some other techniques, computer algorithms and settings for determiningthe degree of sequence identity are for example described in WO04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO98/49185 and GB 2 357 768-A.

Usually, for the purpose of determining the percentage of “sequenceidentity” between two nucleotide sequences in accordance with thecalculation method outlined hereinabove, the nucleotide sequence withthe greatest number of nucleotides will be taken as the “first”nucleotide sequence, and the other nucleotide sequence will be taken asthe “second” nucleotide sequence;

f) For the purposes of comparing two or more amino acid sequences, thepercentage of “sequence identity” between a first amino acid sequenceand a second amino acid sequence may be calculated by dividing [thenumber of amino acid residues in the first amino acid sequence that areidentical to the amino acid residues at the corresponding positions inthe second amino acid sequence] by [the total number of amino acidresidues in the first amino acid sequence] and multiplying by [100%], inwhich each deletion, insertion, substitution or addition of an aminoacid residue in the second amino acid sequence—compared to the firstamino acid sequence—is considered as a difference at a single amino acidresidue (position), i.e. as an “amino acid difference” as defined below.

Alternatively, the degree of sequence identity between two amino acidsequences may be calculated using a known computer algorithm, such asthose mentioned above for determining the degree of sequence identityfor nucleotide sequences, again using standard settings.

Usually, for the purpose of determining the percentage of “sequenceidentity” between two amino acid sequences in accordance with thecalculation method outlined hereinabove, the amino acid sequence withthe greatest number of amino acid residues will be taken as the “first”amino acid sequence, and the other amino acid sequence will be taken asthe “second” amino acid sequence.

Also, in determining the degree of sequence identity between two aminoacid sequences, the skilled person may take into account so-called“conservative” amino acid substitutions, which can generally bedescribed as amino acid substitutions in which an amino acid residue isreplaced with another amino acid residue of similar chemical structureand which has little or essentially no influence on the function,activity or other biological properties of the polypeptide. Suchconservative amino acid substitutions are well known in the art, forexample from WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 andWO 01/09300; and (preferred) types and/or combinations of suchsubstitutions may be selected on the basis of the pertinent teachingsfrom WO 04/037999 as well as WO 98/49185 and from the further referencescited therein.

Such conservative substitutions preferably are substitutions in whichone amino acid within the following groups (a)-(e) is substituted byanother amino acid residue within the same group: (a) small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b)polar, negatively charged residues and their (uncharged) amides: Asp,Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg andLys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys;and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp intoGlu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

Any amino acid substitutions applied to the polypeptides describedherein may also be based on the analysis of the frequencies of aminoacid variations between homologous proteins of different speciesdeveloped by Schulz et al., Principles of Protein Structure,Springer-Verlag, 1978, on the analyses of structure forming potentialsdeveloped by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al., Proc. Nad. Acad Sci.USA 81: 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132,1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986,all incorporated herein in their entirety by reference. Information onthe primary, secondary and tertiary structure of Nanobodies given in thedescription herein and in the general background art cited above. Also,for this purpose, the crystal structure of a VHH domain from a llama isfor example given by Desmyter et al., Nature Structural Biology, Vol. 3,9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); 3,752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999).Further information about some of the amino acid residues that inconventional VH domains form the VH/VL interface and potentialcamelizing substitutions on these positions can be found in the priorart on Nanobodies cited herein;

g) amino acid sequences and nucleic acid sequences are said to be“exactly the same” if they have 100% sequence identity (as definedherein) over their entire length;

h) when comparing two amino acid sequences, the term “amino aciddifference” refers to an insertion, deletion or substitution of a singleamino acid residue on a position of the first sequence, compared to thesecond sequence; it being understood that two amino acid sequences cancontain one, two or more such amino acid differences;i) a nucleic acid sequence or amino acid sequence is considered to be“(in) essentially isolated (form)”—for example, compared to its nativebiological source and/or the reaction medium or cultivation medium fromwhich it has been obtained—when it has been separated from at least oneother component with which it is usually associated in said source ormedium, such as another nucleic acid, another protein/polypeptide,another biological component or macromolecule or at least onecontaminant, impurity or minor component. In particular, a nucleic acidsequence or amino acid sequence is considered “essentially isolated”when it has been purified at least 2-fold, in particular at least10-fold, more in particular at least 100-fold, and up to 1000-fold ormore. A nucleic acid sequence or amino acid sequence that is “inessentially isolated form” is preferably essentially homogeneous, asdetermined using a suitable technique, such as a suitablechromatographical technique, such as polyacrylamide-gelelectrophoresis;j) The term “domain” as used herein generally refers to a globularregion of an antibody chain, and in particular to a globular region of aheavy chain antibody, or to a polypeptide that essentially consists ofsuch a globular region. Usually, such a domain will comprise peptideloops (for example 3 or 4 peptide loops) stabilized, for example, as asheet or by disulfide bonds.k) The term ‘antigenic determinant’ refers to the epitope on the antigenrecognized by the antigen-binding molecule (such as a Nanobody or apolypeptide of the invention) and more in particular by theantigen-binding site of said molecule. The terms “antigenic determinant”and “epitope’ may also be used interchangeably herein.l) An amino acid sequence (such as a Nanobody, an antibody, apolypeptide of the invention, or generally an antigen binding protein orpolypeptide or a fragment thereof) that can bind to, that has affinityfor and/or that has specificity for a specific antigenic determinant,epitope, antigen or protein (or for at least one part, fragment orepitope thereof) is said to be “against” or “directed against” saidantigenic determinant, epitope, antigen or protein.m) The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule or antigen-binding protein (such as a Nanobody or a polypeptideof the invention) molecule can bind. The specificity of anantigen-binding protein can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the KD, the stronger the binding strength between an antigenicdeterminant and the antigen-binding molecule (alternatively, theaffinity can also be expressed as the affinity constant (KA), which is1/KD). As will be clear to the skilled person (for example on the basisof the further disclosure herein), affinity can be determined in amanner known per se, depending on the specific antigen of interest.Avidity is the measure of the strength of binding between anantigen-binding molecule (such as a Nanobody or polypeptide of theinvention) and the pertinent antigen. Avidity is related to both theaffinity between an antigenic determinant and its antigen binding siteon the antigen-binding molecule and the number of pertinent bindingsites present on the antigen-binding molecule. Typically,antigen-binding proteins (such as the Nanobodies and/or polypeptides ofthe invention) will bind with a dissociation constant (KD) of 10⁻⁵ to10⁻¹² moles/liter (M) or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter(M) or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter, and/or withan association constant (K_(A)) of at least 10⁷ M⁻¹, preferably at least10⁸ M⁻¹, more preferably at least 10⁹ M⁻¹, such as at least 10¹² M. AnyK_(D) value greater than 10⁴ M is generally considered to indicatenon-specific binding. Preferably, a Nanobody or polypeptide of theinvention will bind to the desired antigen with an K_(D) less than 500nM, preferably less than 200 nM, more preferably less than 10 nM, suchas less than 500 pM. Specific binding of an antigen-binding protein toan antigen or antigenic determinant can be determined in any suitablemanner known per se, including, for example, Scatchard analysis and/orcompetitive binding assays, such as radioimmunoassays (RIA), enzymeimmunoassays (EIA) and sandwich competition assays, and the differentvariants thereof known per se in the art.n) as further described hereinbelow, the amino acid sequence andstructure of a Nanobody can be considered—without however being limitedthereto—to be comprised of four framework regions or “FR's”, which arereferred to in the art and hereinbelow as “Framework region 1” or “FR1”;as “Framework region 2” or“FR2”; as “Framework region 3” or “FR3”; andas “Framework region 4” or “FR4”, respectively; which framework regionsare interrupted by three complementary determining regions or “CDR's”,which are referred to in the art as “Complementarity Determining Region1” or “CDR1”; as “Complementarity Determining Region 2” or “CDR2”; andas “Complementarity Determining Region 3” or “CDR3”, respectively;o) as also further describe hereinbelow, the total number of amino acidresidues in a Nanobody can be in the region of 110-120, is preferably112-115, and is most preferably 113. It should however be noted thatparts, fragments or analogs (as further described hereinbelow) of aNanobody are not particularly limited as to their length and/or size, aslong as such parts, fragments or analogs meet the further requirementsoutlined hereinbelow and are also preferably suitable for the purposesdescribed herein;p) the amino acid residues of a Nanobody are numbered according to thegeneral numbering for V_(H) domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to V_(HH) domains fromCamelids in the article of Riechmann and Muyldermans, referred to above(see for example FIG. 2 of said reference). According to this numbering,FR1 of a Nanobody comprises the amino acid residues at positions 1-30,CDR1 of a Nanobody comprises the amino acid residues at positions 31-36,FR2 of a Nanobody comprises the amino acids at positions 36-49, CDR2 ofa Nanobody comprises the amino acid residues at positions 50-65, FR3 ofa Nanobody comprises the amino acid residues at positions 66-94, CDR3 ofa Nanobody comprises the amino acid residues at positions 95-102, andFR4 of a Nanobody comprises the amino acid residues at positions103-113. [In this respect, it should be noted that—as is well known inthe art for V_(H) domains and for V_(HH) domains—the total number ofamino acid residues in each of the CDR's may vary and may not correspondto the total number of amino acid residues indicated by the Kabatnumbering (that is, one or more positions according to the Kabatnumbering may not be occupied in the actual sequence, or the actualsequence may contain more amino acid residues than the number allowedfor by the Kabat numbering). This means that, generally, the numberingaccording to Kabat may or may not correspond to the actual numbering ofthe amino acid residues in the actual sequence. Generally, however, itcan be said that, according to the numbering of Kabat and irrespectiveof the number of amino acid residues in the CDR's, position 1 accordingto the Kabat numbering corresponds to the start of FR1 and vice versa,position 36 according to the Kabat numbering corresponds to the start ofFR2 and vice versa, position 66 according to the Kabat numberingcorresponds to the start of FR3 and vice versa, and position 103according to the Kabat numbering corresponds to the start of FR4 andvice versa.].

Alternative methods for numbering the amino acid residues of V_(H)domains, which methods can also be applied in an analogous manner toV_(HH) domains from Camelids and to Nanobodies, are the method describedby Chothia et al. (Nature 342, 877-883 (1989)), the so-called “AbMdefinition” and the so-called “contact definition”. However, in thepresent description, claims and figures, the numbering according toKabat as applied to V_(HH) domains by Riechmann and Muyldermans will befollowed, unless indicated otherwise; and

q) the Figures, Sequence Listing and the Experimental Part/Examples areonly given to further illustrate the invention and should not beinterpreted or construed as limiting the scope of the invention and/orof the appended claims in any way, unless explicitly indicated otherwiseherein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the followingreferences, which are mentioned as general background art: WO 94/04678,WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N.V. and applicant; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 (=EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551 by applicant and the further published patentapplications by applicant;

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In accordance with the terminology used in the above references, thevariable domains present in naturally occurring heavy chain antibodieswill also be referred to as “V_(HH) domains”, in order to distinguishthem from the heavy chain variable domains that are present inconventional 4-chain antibodies (which will be referred to hereinbelowas “V_(H) domains”) and from the light chain variable domains that arepresent in conventional 4-chain antibodies (which will be referred tohereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have anumber of unique structural characteristics and functional propertieswhich make isolated V_(HH) domains (as well as Nanobodies based thereon,which share these structural characteristics and functional propertieswith the naturally occurring V_(HH) domains) and proteins containing thesame highly advantageous for use as functional antigen-binding domainsor proteins. In particular, and without being limited thereto, V_(HH)domains (which have been “designed” by nature to functionally bind to anantigen without the presence of, and without any interaction with, alight chain variable domain) and Nanobodies can function as a single,relatively small, functional antigen-binding structural unit, domain orprotein. This distinguishes the V_(HH) domains from the V_(H) and V_(L)domains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or domains, but need to be combined in some form or another toprovide a functional antigen-binding unit (as in for exampleconventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains andNanobodies as single antigen-binding proteins or as antigen-bindingdomains (i.e. as part of a larger protein or polypeptide) offers anumber of significant advantages over the use of conventional V_(H) andV_(L) domains, scFv's or conventional antibody fragments (such as Fab-or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spacial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   V_(HH) domains and Nanobodies can be expressed from a single        gene and require no post-translational folding or modifications;    -   V_(HH) domains and Nanobodies can easily be engineered into        multivalent and multispecific formats (as further discussed        herein);    -   V_(HH) domains and Nanobodies are highly soluble and do not have        a tendency to aggregate (as with the mouse-derived        antigen-binding domains described by Ward et al., Nature, Vol.        341, 1989, p. 544);    -   V_(HH) domains and Nanobodies are highly stable to heat, pH,        proteases and other denaturing agents or conditions (see for        example Ewert et al, supra);    -   V_(HH) domains and Nanobodies are easy and relatively cheap to        prepare, even on a scale required for production. For example,        V_(HH) domains, Nanobodies and proteins/polypeptides containing        the same can be produced using microbial fermentation (e.g. as        further described below) and do not require the use of mammalian        expression systems, as with for example conventional antibody        fragments;    -   V_(HH) domains and Nanobodies are relatively small        (approximately 15 kDa, or 10 times smaller than a conventional        IgG) compared to conventional 4-chain antibodies and        antigen-binding fragments thereof, and therefore show high(er)        penetration into tissues (including but not limited to solid        tumors and other dense tissues) than such conventional 4-chain        antibodies and antigen-binding fragments thereof;    -   V_(HH) domains and Nanobodies can show so-called cavity-binding        properties (inter alia due to their extended CDR3 loop, compared        to conventional V_(H) domains) and can therefore also access        targets and epitopes not accessible to conventional 4-chain        antibodies and antigen-binding fragments thereof. For example,        it has been shown that V_(HH) domains and Nanobodies can inhibit        enzymes (see for example WO 97/49805; Transue et al., (1998),        supra; Lauwereys et al., (1998), supra.

As mentioned above, the invention generally relates to Nanobodiesdirected against TNF-alpha, as well as to polypeptides comprising oressentially consisting of one or more of such Nanobodies, that can beused for the prophylactic, therapeutic and/or diagnostic purposesdescribed below and in WO 04/041862.

As also mentioned above and further described below, the inventionfurther relates to nucleic acids encoding such Nanobodies andpolypeptides, to methods for preparing such Nanobodies and polypeptides,to host cells expressing or capable of expressing such Nanobodies orpolypeptides, to uses of such Nanobodies, polypeptides, nucleic acids orhost cells, and to compositions comprising such Nanobodies,polypeptides, nucleic acids or host cells.

Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can be obtained (1)by isolating the V_(HH) domain of a naturally occurring heavy chainantibody; (2) by expression of a nucleotide sequence encoding anaturally occurring V_(HH) domain; (3) by “humanization” (as describedbelow) of a naturally occurring V_(HH) domain or by expression of anucleic acid encoding a such humanized V_(HH) domain; (4) by“camelization” (as described below) of a naturally occurring V_(H)domain from any animal species, in particular a species of mammal, suchas from a human being, or by expression of a nucleic acid encoding sucha camelized V_(H) domain; (5) by “camelisation” of a “domain antibody”or “Dab” as described by Ward et al (supra), or by expression of anucleic acid encoding such a camelized V_(H) domain; (6) using syntheticor semi-synthetic techniques for preparing proteins, polypeptides orother amino acid sequences; (7) by preparing a nucleic acid encoding aNanobody using techniques for nucleic acid synthesis, followed byexpression of the nucleic acid thus obtained; and/or (8) by anycombination of the foregoing. Suitable methods and techniques forperforming the foregoing will be clear to the skilled person based onthe disclosure herein and for example include the methods and techniquesdescribed in more detail hereinbelow.

However, according to a specific embodiment, the Nanobodies of theinvention do not have an amino acid sequence that is exactly the same as(i.e. as a degree of sequence identity of 100% with) the amino acidsequence of a naturally occurring V_(H) domain, such as the amino acidsequence of a naturally occurring V_(H) domain from a mammal, and inparticular from a human being.

One particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(HH) domain, but that hasbeen “humanized”, i.e. by replacing one or more amino acid residues inthe amino acid sequence of said naturally occurring V_(HH) sequence byone or more of the amino acid residues that occur at the correspondingposition(s) in a V_(H) domain from a conventional 4-chain antibody froma human being (e.g. indicated above). This can be performed in a mannerknown per se, which will be clear to the skilled person, for example onthe basis of the further description below and the prior art onhumanization referred to herein. Again, it should be noted that suchhumanized Nanobodies of the invention can be obtained in any suitablemanner known per se (i.e. as indicated under points (1)-(8) above) andthus are not strictly limited to polypeptides that have been obtainedusing a polypeptide that comprises a naturally occurring V_(HH) domainas a starting material.

A preferred, but non-limiting humanizing substitution for Nanobodiesbelonging to the 103 P,R,S-group and/or the GLEW-group (as definedherein) is 108Q to 108L.

Another particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(H) domain that has been“camelized”, i.e. by replacing one or more amino acid residues in theamino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe further description below. Reference is also made to WO 94/04678.Such camelization may preferentially occur at amino acid positions whichare present at the V_(H)-V_(L) interface and at the so-called Camelidaehallmark residues (see for example also WO 94/04678), as also mentionedbelow. Preferably, the V_(H) domain or sequence that is used as astarting material or starting point for generating or designing thecamelized Nanobody is preferably a V_(H) sequence from a mammal, morepreferably the V_(H) sequence of a human being. However, it should benoted that such camelized Nanobodies of the invention can be obtained inany suitable manner known per se (i.e. as indicated under points (1)-(8)above) and thus are not strictly limited to polypeptides that have beenobtained using a polypeptide that comprises a naturally occurring V_(H)domain as a starting material.

For example, again as further described below, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes such a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence such that the new nucleotide sequenceencodes a humanized or camelized Nanobody of the invention,respectively, and then expressing the nucleotide sequence thus obtainedin a manner known per se so as to provide the desired Nanobody of theinvention. Alternatively, based on the amino acid sequence of anaturally occurring V_(HH) domain or V_(H) domain, respectively, theamino acid sequence of the desired humanized or camelized Nanobody ofthe invention, respectively, can be designed and then synthesized denovo using techniques for peptide synthesis known per se. Also, based onthe amino acid sequence or nucleotide sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, a nucleotide sequenceencoding the desired humanized or camelized Nanobody of the invention,respectively, can be designed and then synthesized de novo usingtechniques for nucleic acid synthesis known per se, after which thenucleotide sequence thus obtained can be expressed in a manner known perse so as to provide the desired Nanobody of the invention.

Other suitable ways and techniques for obtaining Nanobodies of theinvention and/or nucleotide sequences and/or nucleic acids encoding thesame, starting from (the amino acid sequence of) naturally occurringV_(H) domains or preferably V_(HH) domains and/or from nucleotidesequences and/or nucleic acid sequences encoding the same will be clearfrom the skilled person, and may for example comprising combining one ormore amino acid sequences and/or nucleotide sequences from naturallyoccurring V_(H) domains (such as one or more FR's and/or CDR's) with oneor more amino acid sequences and/or nucleotide sequences from naturallyoccurring V_(HH) domains (such an one or more FR's or CDR's), in asuitable manner so as to provide (a nucleotide sequence or nucleic acidencoding) a Nanobody of the invention.

According to one preferred, but non-limiting aspect of the aspect of theinvention, a Nanobody in its broadest sense can be generally defined asa polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 44    according to the Kabat numbering is E and in which the amino acid    residue at position 45 according to the Kabat numbering is an R;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   i) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:-   ii) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   iii) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which-   iv) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In particular, a Nanobody against TNF-alpha according to the inventionmay have the structure:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   i) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:-   ii) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   iii) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which-   iv) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In particular, according to one preferred, but non-limiting aspect ofthe aspect of the invention, a Nanobody can generally be defined as apolypeptide comprising an amino acid sequence that is comprised of fourframework regions/sequences interrupted by three complementaritydetermining regions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of G, E, D, G, Q, R,    S, L; and is preferably chosen from the group consisting of G, E or    Q; and-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R; and-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    or in which:-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q; and-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R; and-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    or in which:-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q; and-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R; and-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S; and-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of G, E, D, G, Q, R,    S, L; and is preferably chosen from the group consisting of G, E or    Q;    and in which:-   ii) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R;    and in which:-   iii) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;    and in which-   iv) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and in which:-   v) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q;    and in which:-   ii) the amino acid residue at position 45 according to the Kabat    numbering is R;    and in which:-   iii) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;    and in which:-   iv) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and in which:-   vi) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q;    and in which:-   ii) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R;    and in which:-   iii) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S;    and in which:-   iv) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which:-   v) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

Two particularly preferred, but non-limiting groups of the Nanobodies ofthe invention are those according to a) above; according to I) to a-4)above; according to b) above; according to b-1) to b-4) above; accordingto c) above; and/or according to c-1) to c-4) above, in which;

-   a) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    below) and the amino acid residue at position 108 is Q;    or in which:-   b) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE (SEQ ID NO: 437) or KQRE (SEQ ID    NO: 438) (or a KERE-like sequence) and the amino acid residue at    position 108 is Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    below) and the amino acid residue at position 108 is Q;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE (SEQ ID NO: 437) or KQRE (SEQ ID    NO: 438) (or a KERE-like sequence) and the amino acid residue at    position 108 is Q or L, and is preferably Q;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In the Nanobodies of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KERE(SEQ ID NO: 437) or KQRE (SEQ ID NO: 438), the amino acid residue atposition 37 is most preferably F. In the Nanobodies of the invention inwhich the amino acid residues at positions 44-47 according to the Kabatnumbering form the sequence GLEW, the amino acid residue at position 37is chosen from the group consisting of Y, H, I, V or F, and is mostpreferably F.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the Nanobodiesof the invention can generally be classified is on the basis of thefollowing three groups:

-   a) The “GLEW-group”: Nanobodies with the amino acid sequence GLEW at    positions 44-47 according to the Kabat numbering and Q at position    108 according to the Kabat numbering. As further described herein,    Nanobodies within this group usually have a V at position 37, and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. The GLEW group also comprises some GLEW-like sequences    such as those mentioned in Table 2 below;-   b) The “KERE-group”: Nanobodies with the amino acid sequence KERE    (SEQ ID NO: 437) or KQRE (SEQ ID NO: 438) or at positions 43-46    according to the Kabat numbering and Q or L at position 108    according to the Kabat numbering. As further described herein,    Nanobodies within this group usually have a F at position 37, an L    or F at position 47; and can have a W, P, R or S at position 103,    and preferably have a W at position 103;-   c) The “103 P, R, S-group”: Nanobodies with a P R or S at    position 103. These Nanobodies can have either the amino acid    sequence GLEW (SEQ ID NO: 439) at positions 44-47 of the Kabat    numbering or the amino acid sequence KERE (SEQ ID NO: 437) or KQRE    (SEQ ID NO: 438) at positions 43-46 according to the Kabat    numbering, the latter most preferably in combination with an F at    position 37 and an L or an F at position 47 (as defined for the    KERE-group); and can have Q or L at position 108 according to the    Kabat numbering, and preferably have Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined above, and arepreferably as defined according to one of the preferred definitionsabove, and are more preferably as defined according to one of the morepreferred definitions above.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the KERE-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined above, and arepreferably as defined according to one of the preferred definitionsabove, and are more preferably as defined according to one of the morepreferred definitions above.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the 103 P, R, S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined above,and are preferably as defined according to one of the preferreddefinitions above, and are more preferably as defined according to oneof the more preferred definitions above.

Also, more generally and in addition to the 108Q, 43E/44R and 103P,R,Sresidues mentioned above, the Nanobodies of the invention can contain,at one or more positions that, in a conventional V_(H) domain, wouldform (part of) the V_(H)/V_(L) interface, contain one or more amino acidresidues that are more highly charged than the amino acid residues thatnaturally occur at the same position(s) in the corresponding naturallyoccurring V_(H) or V_(HH) domain, and in particular one or more chargedamino acid residues (as mentioned in Table 1).

Such substitutions include, but are not limited to the GLEW-likesequences mentioned in Table 2 below; as well as the substitutions thatare described in the International Application WO 00/29004 for so-called“microbodies”, e.g. a Q at position 108 and KLEW at positions 44-47.

In some embodiments of the Nanobodies of the invention, the amino acidresidue at position 83 is chosen from the group consisting of L, M, S, Vand W; and is preferably L.

Also, in some embodiments of the Nanobodies of the invention, the aminoacid residue at position 83 is chosen from the group consisting of R, K,N, E, I and Q; and is most preferably either K or E (for Nanobodiescorresponding to naturally occurring V_(HH) domains) or R (for“humanized” Nanobodies, as described below). The amino acid residue atposition 84 in some embodiments is chosen from the group consisting ofP, A, R, S, D and V, and is most preferably P (for Nanobodiescorresponding to naturally occurring V_(HH) domains) or R (for“humanized” Nanobodies, as described below).

Furthermore, in some embodiments of the Nanobodies of the invention, theamino acid residue at position 104 is chosen from the group consistingof G and D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the Nanobodies are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human VH domain, VH3, aresummarized in Table 2.

Some especially preferred combinations of these Hallmark Residues asoccur in naturally occurring V_(HH) domains are mentioned in Table 3.For comparison, the corresponding amino acid residues of the humanV_(H)3 called DP-47 have been indicated in italics.

TABLE 2 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues 11 L, V; L, M, S, V, W; preferably L predominantly L 37 V, I,F; F⁽¹⁾, Y, H, I or V, preferably F⁽¹⁾ or Y usually V  44⁽⁸⁾ G G⁽²⁾,E⁽³⁾, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾ orE⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S or Y; preferably W⁽²⁾ ,L⁽¹⁾, F⁽¹⁾ or R 83 R or K; usually R R, K⁽⁵⁾, N, E⁽⁵⁾, I, M or Q;preferably K or R; most preferably K 84 A, T, D; P⁽⁵⁾, A, L, R, S, D, V;preferably P predominantly A 103  W W⁽⁴⁾, P⁽⁶⁾ , R⁽⁶⁾, S; preferably W104  G G or D; preferably G 108  L, M or T; Q, L⁽⁷⁾ or R; preferably Qor L⁽⁷⁾ predominantly L Notes: ⁽¹⁾In particular, but not exclusively, incombination with KERE (SEQ ID NO: 437) or KQRE (SEQ ID NO: 438) atpositions 43-46. ⁽²⁾Usually as GLEW (SEQ ID NO: 439) at positions 44-47.⁽³⁾Usually as KERE or KQRE at positions 43-46, e.g. as KEREL (SEQ ID NO:440), KEREF (SEQ ID NO: 441), KQREL (SEQ ID NO: 442), KQREF (SEQ ID NO:443) or KEREG (SEQ ID NO: 444) at positions 43-47. Alternatively, alsosequences such as TERE (SEQ ID NO: 445) (for example TEREL (SEQ ID NO:446)), KECE (SEQ ID NO: 447) (for example KECEL (SEQ ID NO: 448) orKECER (SEQ ID NO: 449)), RERE (SEQ ID NO: 450) (for example REREG (SEQID NO: 451)), QERE (SEQ ID NO: 452) (for example QEREG (SEQ ID NO:453)), KGRE (SEQ ID NO: 454) (for example KGREG (SEQ ID NO: 455)), KDRE(SEQ ID NO: 456) (for example KDREV (SEQ ID NO: 457)) are possible. Someother possible, but less preferred sequences include for example DECKL(SEQ ID NO: 458) and NVCEL (SEQ ID NO: 459). ⁽⁴⁾With both GLEW atpositions 44-47 and KERE or KQRE at positions 43-46. ⁽⁵⁾Often as KP orEP at positions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾Inparticular, but not exclusively, in combination with GLEW at positions44-47. ⁽⁷⁾With the proviso that when positions 44-47 are GLEW, position108 is always Q. ⁽⁸⁾The GLEW group also contains GLEW-like sequences atpositions 44-47, such as for example GVEW (SEQ ID NO: 460), EPEW (SEQ IDNO: 461), GLER (SEQ ID NO: 462), DQEW (SEQ ID NO: 463), DLEW (SEQ ID NO:464), GIEW (SEQ ID NO: 465), ELEW (SEQ ID NO: 466), GPEW (SEQ ID NO:467), EWLP (SEQ ID NO: 468), GPER (SEQ ID NO: 469), GLER (SEQ ID NO:470).

TABLE 3 Some preferred combinations of Hallmark Residues in naturallyoccurring Nanobodies. For humanization of these combinations, referenceis made to the specification. 11 37 44 45 47 83 84 103 104 108 DP-47(human) M V G L W R A W G L “KERE” group L F E R L K P W G Q L F E R F EP W G Q L F E R F K P W G Q L Y Q R L K P W G Q L F L R V K P Q G Q L FQ R L K P W G Q L F E R F K P W G Q “GLEW” group L V G L W K S W G Q M VG L W K P R G Q

In the Nanobodies, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables 4-7mention some non-limiting residues that can be present at each position(according to the Kabat numbering) of the FR1, FR2, FR3 and FR4 ofnaturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a Nanobody) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering Chothia (supra) that the residues at thesepositions already form part of CDR1.)

In Tables 4-7, some of the non-limiting residues that can be present ateach position of a human V_(H)3 domain have also been mentioned. Again,for each position, the amino acid residue that most frequently occurs ateach position of a naturally occurring human V_(H)3 domain is indicatedin bold; and other preferred amino acid residues have been underlined.

TABLE 4 Non-limiting examples of amino acid residues in FR1 (for thefootnotes, see the footnotes to Table 2) Amino acid residue(s): Pos.Human V_(H)3 Camelid V_(HH)'s 1 E, Q Q, A, E, D, H, R 2 V V, A, E, G, L,M, Q 3 Q Q, K, E, H, P, R, Y 4 L L, F, P, R, V 5 V, L Q, E, L, V, M, P,A, I 6 E E, D, Q, A, H 7 S, T S, F, H 8 G, R G, A, R 9 G G, E 10 G, V G,D, R, A, E, N, T, V 11 Hallmark residue: L, M, S, V, W, F, N, P, T, Y;preferably L 12 V, I V, A, G, M 13 Q, K, R Q, E, K, D, G, A, H, L, N, P,R, T 14 P A, Q, A, G, P, T, V, E, F, I, N, S 15 G G 16 G, R G, A, E, D,N, P, R, S, V, W 17 S S, F, T, N, P, A, C 18 L L, V, M, Q, R 19 R, K R,K, L, N, S, T, A, F, G, I, M, Q 20 L L, F, I, V, M, S 21 S S, F, T, G,H, P, A 22 C C 23 A, T A, D, P, S, T, V, E, G, I, L, Q, R 24 A A, I, S,T, V, C, E, F, G, L, N, P, Q, Y 25 S S, A, F, P, T, L, V 26 G G, D, E,R, S, V, A, I, M, P, T 27 F S, F, R, L, P, G, N, A, D, E, H, I, K, M, Q,T, V, Y 28 T N, T, E, D, S, I, R, A, G, R, F, Y, L, M, P, V 29 F, V F,L, D, S, I, G, V, A, E, P, T, Y 30 S, D, G N, S, E, G, A, D, M, T, H, I,P, R, V, W

TABLE 5 Non-limiting examples of amino acid residues in FR2 (for thefootnotes, see the footnotes to Table 2) Amino acid residue(s): Pos.Human V_(H)3 Camelid V_(HH)'s 36 W W 37 Hallmark residue: F⁽¹⁾, Y, H, I,A, L, P, S or V preferably F⁽¹⁾ or Y 38 R R 39 Q Q, H, P, R, A, D, G, L,E 40 A A, F, G, P, T, V, I, L, N, R, S, Y 41 P, S, T P, A, L, S, I, Q, T42 G G, E, D, R, T, V 43 K K, D, E, N, Q, R, T, V, A, L, M, S 44Hallmark residue: G⁽²⁾, E⁽³⁾, D, Q, R, S, L, A, F, K, M, N, P, V, W, Y;preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾ or E⁽³⁾. 45Hallmark residue: L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V, D, E, G, H, K, T;preferably L⁽²⁾ or R⁽³⁾ 46 E, V E, D, K, Q, V, A, G, N 47Hallmark residue: W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, D, E, H, K, Q,T, V or Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L, A, C, E, F, G,H, M, P, Q, R, S, T, V, W, Y 49 S, A, G A, S, A, G, T, V, D, E, I, L, Q,R, Y

TABLE 6 Non-limiting examples of amino acid residues in FR3 (for thefootnotes, see the footnotes to Table 2) Amino acid residue(s): Pos.Human V_(H)3 Camelid V_(HH)'s 66 R R 67 F F, L, V, A, D, I, S, Y 68 T T,A, S, D, F, G, I, K, N 69 I I, M, V, A, F, L, R, S, T 70 S S, A, F, E,G, K, P, T, V 71 R R, G, I, K, Q, S, T, W, A, F, L, M, N 72 D, E D, E,G, N, V, A, H, I, L, Q, S, T 73 N, D, G N, D, F, I, K, S, T, Y, A, G, H,L, M, R, V 74 A, S A, D, G, N, P, S, T, F, H, I, L, R, V, Y 75 K K, A,E, K, L, N, Q, R, D, G, I, M, S, T, V, W 76 N, S N, D, K, R, S, T, Y, E,G, H, I, Q 77 S, T, I T, A, E, I, M, S, K, L, N, R, V 78 L, A V, L, A,F, G, I, M, E, N, Q, R, S, T, W 79 Y, H Y, A, D, F, H, S, T, C, E, I, L,N, V, W 80 L L, F, V, M 81 Q Q, E, R, T, G, H, I, K, L, M, N 82 M M, I,L, V, G, P, T 82a N, G N, D, G, H, S, T, A, E, I, K, R, V 82b S S, N, D,G, R, A, C, E, F, I, K, M, P, T, V 82c L L, P, M, T, V 83Hallmark residue: R, K⁽⁵⁾, N, E⁽⁵⁾, I, M, A, D, G, L, Q, S, T or Q;preferably K or R; most preferably K 84 Hallmark residue: P⁽⁵⁾, A, L, R,S, D, V, F, G, H, N, T, Y; preferably P 85 E, G E, D, G, Q, A, N, R, V,Y 86 D D, E, F, Y 87 T, M T, S, A, C, M 88 A A, G, S, D, L, N, P 89 V, LV, A, D, I, L, M, N, R, T, E, F, S 90 Y Y, F, E, H, N 91 Y, H Y, D, F,H, L, S, T, V, C, I, N, R, W 92 C C 93 A, K, T A, N, G, H, K, R, S, T,V, Y, E, F, I, L, M, Q 94 K, R, T A, V, C, F, G, I, L, R, S, D, E, K, M,N, P, Q, T, W, Y T or K;

TABLE 7 Non-limiting examples of amino acid residues in FR4 (for thefootnotes, see the footnotes to Table 2) Amino acid residue(s): Pos.Human V_(H)3 Camelid V_(HH)'s 103 Hallmark residue: W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S,F, G, K, L, N, Q, V, Y; preferably W 104 Hallmark residue: G, A, R, S, Tor D; preferably G 105 Q, R Q, E, K, P, R, G, H, L, S, V 106 G G 107 TT, A, I, N, P 108 Hallmark residue: Q, L⁽⁷⁾, E, H, N, P, T or R;preferably Q or L⁽⁷⁾ 109 V V 110 T T, I, A 111 V V, A, I, G 112 S S, F,A, L, P, T, Y 113 S S, A, L, P, F, T

Thus, in another preferred, but not limiting aspect, a Nanobody of theinvention can have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the Hallmark residues are as defined above;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which: and in which

-   i) FR1 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 1] [1] QVQLQESGGG X VQAGGSLRLSCAASG [26]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 4; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 4; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and in which:

-   ii) FR2 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 2] [36] W X RQAPGK XX E X VA [49]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 5; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 5; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and in which:

-   iii) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 3] [66] RFTISRDNAKNTVYLQMNSL XX EDTAVYYCAA [94]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 7; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 7; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and in which:

-   iv) FR4 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 4] [103]  XX QGT X VTVSS [113]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and in which:

-   v) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above;    in which the Hallmark Residues are indicated by “X” and are as    defined hereinabove and in which the numbers between brackets refer    to the amino acid positions according to the Kabat numbering.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:and in which

-   i) FR1 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 5] [1] QVQLQESGGG L VQAGGSLRLSCAASG [26]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 4; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residue at position is as indicated in the        sequence above;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 4; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residue at position is as indicated in the        sequence above;        and in which:

-   ii) FR2 is chosen from the group consisting of the amino acid    sequences:

[SEQ ID NO: 6] [36] W F RQAPGK ER E L VA [49] [SEQ ID NO: 7] [36] W FRQAPGK ER E F VA [49] [SEQ ID NO: 8] [36] W F RQAPGK ER E G A [49] [SEQID NO: 9] [36] W F RQAPGK QR E L VA [49] [SEQ ID NO: 10] [36] W F RQAPGKQR E F VA [49] [SEQ ID NO: 11] [36] W Y RQAPGK GL E W A [49]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 5; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in each of the sequences above;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 5; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in each of the sequences above;        and in which:

-   iii) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 12] [66] RFTISRDNAKNTVYLQMNSL KP EDTAVYYCAA [94]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 83 and 84 are as        indicated in each of the sequences above;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 83 and 84 are as        indicated in each of the sequences above;        and in which:

-   iv) FR4 is chosen from the group consisting of the amino acid    sequences:

[SEQ ID NO: 13] [103]  WG QGT Q VTVSS [113]

[SEQ ID NO: 14] [103]  WG QGT L VTVSS [113]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequence;

in which

-   -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 103, 104 and 108 are as        indicated in each of the sequences above;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 103, 104 and 108 are as        indicated in each of the sequences above;        and in which:

-   v) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:and in which

-   i) FR1 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 5] [1] QVQLQESGGG L VQAGGSLRLSCAASG [26]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 4; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residue at position is as indicated in the        sequence above; and in which:

ii) FR2 is chosen from the group consisting of the amino acid sequences:

[SEQ ID NO: 6] [36] W F RQAPGK ER E L VA [49] [SEQ ID NO: 7] [36] W FRQAPGK ER E F VA [49] [SEQ ID NO: 8] [36] W F RQAPGK ER E G A [49] [SEQID NO: 9] [36] W F RQAPGK QR E L VA [49] [SEQ ID NO: 10] [36] W F RQAPGKQR E F VA [49]

and/or from the group consisting of amino acid sequences that have 2 oronly 1 “amino acid difference(s)” (as defined herein) with one of theabove amino acid sequences, in which:

-   -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 5; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in each of the sequences above;        and in which:

-   iii) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 12] [66] RFTISRDNAKNTVYLQMNSL KP EDTAVYYCAA [94]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 83 and 84 are as        indicated in each of the sequences above;        and in which:

-   iv) FR4 is chosen from the group consisting of the amino acid    sequences:

[SEQ ID NO: 13] [103]  WG QGT Q VTVSS [113] [SEQ ID NO: 14] [103]  WGQGT L VTVSS [113]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 7; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 103, 104 and 108 are as        indicated in each of the sequences above;        and in which:

-   v) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which: and in which

-   i) FR1 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 5] [1] QVQLQESGGG L VQAGGSLRLSCAASG [26]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 4; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residue at position is as indicated in the        sequence above;        and in which:

-   ii) FR2 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 11] [36] W Y RQAPGK GL E W A [49]

-   -   and/or from the group consisting of amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 5; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in each of the sequences above;        and in which:

-   iii) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 12] [66] RFTISRDNAKNTVYLQMNSL KP EDTAVYYCAA [94]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 83 and 84 are as        indicated in each of the sequences above;        and in which:

-   iv) FR4 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 13] [103]  WG QGT Q VTVSS [113]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 7; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   (3) the Hallmark residues at positions 103, 104 and 108 are as        indicated in each of the sequences above;        and in which:

-   v) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

Some other framework sequences that can be present in the Nanobodies ofthe invention can be found in the European patent EP 656 946 mentionedabove (see for example also the granted equivalent U.S. Pat. No.5,759,808).

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which: and in which

-   i) FR1 is chosen from the group consisting of the FR1 sequences    present in the Nanobodies of SEQ ID NO's 52 to 60, SEQ ID NO's 76 to    86 or SEQ ID NO's 95 to 99, and in particular in the humanized    Nanobodies of SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99,    -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of said FR1 sequences; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 4; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR1 sequence; and    -   (3) the Hallmark residue at position is as indicated in said FR1        sequence; and/or from the group consisting of amino acid        sequences that have 3, 2 or only 1 “amino acid difference(s)”        (as defined herein) with one of said FR1 sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 4; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR1 sequence; and    -   (3) the Hallmark residue at position is as indicated in said FR1        sequence;        and in which:-   ii) FR2 is chosen from the group consisting of the FR2 sequences    present in the Nanobodies of SEQ ID NO's 52 to 60, SEQ ID NO's 76 to    86 or SEQ ID NO's 95 to 99, and in particular in the humanized    Nanobodies of SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99,    -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of said FR2 sequences; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 5; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR2 sequence; and    -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in said FR2 sequence;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of said FR2 sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 5; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR2 sequence; and    -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in said FR2 sequence;        and in which:-   iii) FR3 is chosen from the group consisting of the FR3 sequences    present in the Nanobodies of SEQ ID NO's 52 to 60, SEQ ID NO's 76 to    86 or SEQ ID NO's 95 to 99, and in particular in the humanized    Nanobodies of SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99,    -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of said FR3 sequences; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR3 sequence; and    -   (3) the Hallmark residues at positions 83 and 84 are as        indicated in said FR3 sequence;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of said FR3 sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR3 sequence; and    -   (3) the Hallmark residues at positions 83 and 84 are as        indicated in said FR3 sequence;        and in which:-   iv) FR4 is chosen from the group consisting of the FR4 sequences    present in the Nanobodies of SEQ ID NO's 52 to 60, SEQ ID NO's 76 to    86 or SEQ ID NO's 95 to 99, and in particular in the humanized    Nanobodies of SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99,    -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of said FR4 sequences; in which    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR4 sequence; and    -   (3) the Hallmark residues at positions 103, 104 and 108 are as        indicated in said FR4 sequence;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of said FR4 sequences, in which:    -   (1) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table 6; and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR4 sequence; and    -   (3) the Hallmark residues at positions 103, 104 and 108 are as        indicated in said FR4 sequence;        and in which:-   v) CDR1, CDR2 and CDR3 are as defined above, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

Some particularly preferred Nanobodies of the invention can be chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's 52to 60, SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99, and in particularin the humanized Nanobodies of SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to99 or from the group consisting of amino acid sequences that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity (as defined herein) withone of the amino acid sequences of SEQ ID NO's 52 to 60, SEQ ID NO's 76to 86 or SEQ ID NO's 95 to 99; in which

-   -   (1) the Hallmark residues can be as indicated in Table 2 above;    -   (2) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Tables 4-7; and/or    -   (3) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s).

Some even more particularly preferred Nanobodies of the invention can bechosen from the group consisting of the amino acid sequences of SEQ IDNO's 52 to 60, SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99, and inparticular in the humanized Nanobodies of SEQ ID NO's 76 to 86 or SEQ IDNO's 95 to 99 or from the group consisting of amino acid sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity (as definedherein) with one of the amino acid sequences of SEQ ID NO's 52 to 60,SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99; in which

-   -   (1) the Hallmark residues are as indicated in the pertinent        sequence chosen from SEQ ID NO's 52 to 60, SEQ ID NO's 76 to 86        or SEQ ID NO's 95 to 99;    -   (2) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Tables 4-7; and/or    -   (3) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the pertinent sequence chosen from SEQ ID NO's 52 to        60, SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99.

Some of the most preferred Nanobodies of the invention can be chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's 52to 60, SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99, and in particularfrom the humanized Nanobodies of SEQ ID NO's 76 to 86 or SEQ ID NO's 95to 99.

As will be clear from the above, the term Nanobodies of the invention asused herein in its broadest sense also comprises natural or syntheticmutants, variants, alleles, analogs and orthologs (hereinbelowcollectively referred to as “analogs”) of the Nanobodies mentioned inthe SEQ ID NO's 52 to 60, SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99.

Generally, such analogs can for example comprise homologous sequences,functional portions, or a functional portion of a homologous sequence(as further defined below) of a Nanobody. Generally, in such analogs,each amino acid residue (other than the Hallmark Residue) in each of theframework regions can be replaced by any other amino acid residue,provided that the total degree of sequence identity of the frameworkregions remains as defined above. Preferably, however, in such analogs:

-   -   one or more amino acid residues in the above framework sequences        are replaced by one or more amino acid residues that naturally        occur at the same position in a naturally occurring V_(HH)        domain. Some examples of such substitutions are mentioned in        Tables 4-7 above;        and/or:    -   one or amino acid residues in the above framework sequences are        replaced by one or more amino acid residues that can be        considered a “conservative” amino acid substitution, as        described hereinabove;        and/or:    -   one or amino acid residues in the above framework sequences are        replaced by one or more amino acid residues that naturally occur        at the same position in a naturally occurring V_(H) domain of a        human being. This is generally referred to as “humanization” of        the naturally occurring V_(HH)/Nanobody in general and of said        position in particular, and will be discussed in more detail        hereinbelow;        and:    -   positions for which only one amino acid residue is mentioned for        both the V_(H) domain and the V_(HH) domain in Tables 4-7 above        are preferably not replaced.

Also, although generally less preferred, in such analogs, one or moreamino acid residues may be deleted from the framework regions and/orinserted into the framework regions (optionally in addition to one ormore amino acid substitutions as mentioned above), provided that thetotal degree of sequence identity of the framework regions remains asdefined above. The Hallmark residues should not be deleted. Also, mostpreferably, amino acid residues for which only one amino acid residue ismentioned for both the V_(H) domain and the V_(HH) domain in Tables 4-7above are preferably not deleted.

Preferably, such analogs should be such that they still can bind to,have affinity for and/or have specificity for TNF-alpha, i.e. with anaffinity and/or a specificity which is at least 10%, preferably at least50%, more preferably at least 70%, even more preferably at least 80%,such as at least 90%, at least 95%, at least 99% or more, of theaffinity and/or specificity of at least one of the Nanobodies of SEQ IDNo's SEQ ID NO's 52 to 60, SEQ ID NO's 76 to 86 or SEQ ID NO's 95 to 99,as determined using a suitable assay, for example an assay to determinebinding of the analog to TNF, and in particular one of the assays asused in the Examples below.

Generally, such analogs can for example be obtained by providing anucleic acid that encodes a naturally occurring V_(HH) domain, changingthe codons for the one or more amino acid residues that are to behumanized into the codons for the corresponding human amino acidresidue(s), expressing the nucleic acid/nucleotide sequence thusobtained in a suitable host or expression system; and optionallyisolating and/or purifying the analog thus obtained to provide saidanalog in essentially isolated form (as defined hereinabove). This cangenerally be performed using methods and techniques known per se, whichwill be clear to the skilled person, for example from the handbooks andreferences cited herein and/or from the further description hereinbelow.Alternatively, and for example, a nucleic acid encoding an analog can besynthesized in a manner known per se (for example using an automatedapparatus for synthesizing nucleic acid sequences with a predefinedamino acid sequence) and can be expressed in a suitable host orexpression system, upon which the analog thus obtained can optionally beisolated and/or purified so as to provide said analog in essentiallyisolated form (as defined hereinabove). Another way to provide theanalogs involves chemical synthesis of the pertinent amino acid sequenceusing techniques for peptide synthesis known per se, such as thosementioned hereinbelow.

It will be also generally be clear to the skilled person that Nanobodies(including analogs thereof) can also be prepared starting from humanV_(H) sequences (i.e. amino acid sequences or the correspondingnucleotide sequences), such as for example human V_(H)3 sequences suchas DP-47, DP-51, DP-54 or DP-29, by changing one or more amino acidresidues in the amino acid sequence of said human V_(H) domain, so as toprovide an amino acid sequence that has (a) a Q at position 108; and/or(b) E at position 44 and/or R at position 45, and preferably E atposition 44 and R at position 45; and/or (c) P, R or S at position 103,as described above. Again, this can generally be performed using thevarious methods and techniques referred to in the previous paragraph,using an amino acid sequence and/or nucleotide sequence for a humanV_(H) domain as a starting point.

The term Nanobodies as used herein in its broadest sense also comprisesparts or fragments of the Nanobodies (including analogs) of theinvention as defined above, which can again be as further describedbelow.

Generally, parts or fragments of the Nanobodies and/or analogs haveamino acid sequences in which, compared to the amino acid sequence ofthe corresponding full length Nanobody or analog, one or more of theamino acid residues at the N-terminal end, one or more amino acidresidues at the C-terminal end, one or more contiguous internal aminoacid residues, or any combination thereof, have been deleted and/orremoved. It is also possible to combine one or more of such parts orfragments to provide a Nanobody of the invention.

Preferably, the amino acid sequence of a Nanobody that comprises one ormore parts or fragments of a full length Nanobody and/or analog shouldhave a degree of sequence identity of at least 50%, preferably at least60%, more preferably at least 70%, such as at least 80%, at least 90% orat least 95%, with the amino acid sequence of the corresponding fulllength Nanobody.

Also, the amino acid sequence of a Nanobody that comprises one or moreparts or fragments of a full length Nanobody and/or analog is preferablysuch that is comprises at least 10 contiguous amino acid residues,preferably at least 20 contiguous amino acid residues, more preferablyat least 30 contiguous amino acid residues, such as at least 40contiguous amino acid residues, of the amino acid sequence of thecorresponding full length Nanobody.

Generally, such parts or fragments of the Nanobodies of the inventionwill have amino acid sequences in which, compared to the amino acidsequence of the corresponding full length Nanobody of the invention, oneor more of the amino acid residues at the N-terminal end, one or moreamino acid residues at the C-terminal end, one or more contiguousinternal amino acid residues, or any combination thereof, have beendeleted and/or removed. It is also possible to combine one or more ofsuch parts or fragments to provide a Nanobody of the invention.

According to one preferred embodiment, a fragment as used hereincomprises at least one of the CDR's present in a full-sized Nanobody ofthe invention, preferably at least two of the CDR's present in afull-sized Nanobody of the invention, more preferably at least CDR2 andCDR3 present in a full-sized Nanobody of the invention, such as forexample all three CDR's present in a full-sized Nanobody of theinvention.

According to another particularly preferred, but non-limitingembodiment, such a part or fragment comprises at least FR3, CDR3 and FR4of the corresponding full length Nanobody of the invention, i.e. as forexample described in the International application WO 03/050531 (Lasterset al.).

Preferably, such parts or fragments should be such that they still canbind to, have affinity for and/or have specificity for TNF-alpha, i.e.with an affinity and/or a specificity which is at least 10%, preferablyat least 50%, more preferably at least 70%, even more preferably atleast 80%, such as at least 90%, at least 95%, at least 99% or more, ofthe affinity and/or specificity of the corresponding full-sized Nanobodyof the invention, for example an assay to determine binding of theanalog to TNF, and in particular one of the assays as used in theExamples below.

From the description hereinabove, it will be clear that the amino acidsequences of the Nanobodies used herein differ at at least one aminoacid position in at least one of the framework regions from the aminoacid sequences of naturally occurring V_(H) domains, such as the aminoacid sequences of naturally occurring V_(H) domains of antibodies fromhuman beings. In particular, it will be clear that the amino acidsequences of the Nanobodies used herein differ at at least one of theHallmark Residues from amino acid sequences of naturally occurring V_(H)domains, such as the amino acid sequences of naturally occurring V_(H)domains from antibodies from Camelids and/or human beings.

Thus, according to one specific embodiment, a Nanobody of the inventionhas an amino acid sequence that differs at at least one amino acidposition in one of the framework regions from the amino acid sequence ofa naturally occurring V_(H) domain. According to a more specific, butnon-limiting embodiment of the invention, a Nanobody of the inventionhas an amino acid sequence that differs at at least one of the Hallmarkresidues from the amino acid sequence of a naturally occurring V_(H)domain.

From the description hereinabove, it will also be clear that the aminoacid sequences of the some of the Nanobodies of the invention, such asthe humanized Nanobodies of the invention, will differ at at least oneamino acid position in at least one of the framework regions (i.e.either at the position of a Hallmark residue or at another position)from the amino acid sequences of naturally occurring V_(HH) domains.Thus, according to one specific, but non-limiting embodiment, a Nanobodyof the invention has an amino acid sequence that differs at at least oneamino acid position in one of the framework regions from the amino acidsequence of a naturally occurring V_(HH) domain. According to a morespecific, but non-limiting embodiment of the invention, a Nanobody ofthe invention has an amino acid sequence that differs at at least one ofthe Hallmark residues from the amino acid sequence of a naturallyoccurring V_(HH) domain.

The invention in its broadest sense also comprises derivatives of theNanobodies of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.g.enzymatical) modification, of the Nanobodies of the invention and/or ofone or more of the amino acid residues that form the Nanobodies of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the Nanobody sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the Nanobody of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that that increase the half-life, the solubilityand/or the absorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or the reducing immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

With regard to pegylation, its should be noted that generally, theinvention also encompasses any Nanobody of the invention and/orpolypeptide of the invention that has been pegylated at one or moreamino acid positions, preferably in such a way that said pegylationeither (1) increases the half-life in vivo; (2) reduces immunogenicity;(3) provides one or more further beneficial properties known per se forpegylation; (4) does not essentially affect the affinity of the Nanobodyand/or polypeptide for TNF-alpha (e.g. does not reduce said affinity bymore than 90%, preferably not by more than 50%, and more preferably notby more than 10%, as determined by a suitable assay, such as thosedescribed in the Examples below); and/or (4) does not affect any of theother desired properties of the Nanobodies and/or polypeptides of theinvention. Suitable PEG-groups and methods for attaching them, eitherspecifically or non-specifically, will be clear to the skilled person.Suitable kits and reagents for such pegylation can for example beobtained from Nektar (CA, USA).

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to,fluorescent labels (such as fluorescein, isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, andfluorescamine and fluorescent metals such as ¹⁵²Eu or others metals fromthe lanthanide series), phosphorescent labels, chemiluminescent labelsor bioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metals chelates ormetallic cations (for example metallic cations such as ^(99m)Tc, ¹²³I,¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metalliccations that are particularly suited for use in in vivo, in vitro or insitu diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe), as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels will beclear to the skilled person, and for example include moieties that canbe detected using NMR or ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, EIA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethyl-enetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

As mentioned above, the invention also relates to proteins orpolypeptides comprising at least one V_(HH) domain (i.e. as identifiedusing the methods of the invention) or at least one Nanobody basedthereon.

According to one non-limiting embodiment of the invention, such apolypeptide of the invention essentially consists of a Nanobody. By“essentially consist of” is meant that the amino acid sequence of thepolypeptide of the invention either is exactly the same as the aminoacid sequence of a Nanobody (as mentioned above) or corresponds to theamino acid sequence of a Nanobody in which a limited number of aminoacid residues, such as 1-10 amino acid residues and preferably 1-6 aminoacid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, have beenadded to the amino terminal end, to the carboxy terminal end, or both tothe amino terminal end and to the carboxy terminal end of the amino acidsequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, such amino acidresidues:

-   a) can comprise an N-terminal Met residue, for example as result of    expression in a heterologous host cell or host organism.-   b) may form a signal sequence or leader sequence that directs    secretion of the Nanobody from a host cell upon synthesis. Suitable    secretory leader peptides will be clear to the skilled person, and    may be as further described herein. Usually, such a leader sequence    will be linked to the N-terminus of the Nanobody, although the    invention in its broadest sense is not limited thereto;-   c) may form a sequence or signal that allows the Nanobody to be    directed towards and/or to penetrate or enter into specific organs,    tissues, cells, or parts or compartments of cells, and/or that    allows the Nanobody to penetrate or cross a biological barrier such    as a cell membrane, a cell layer such as a layer of epithelial    cells, a tumor including solid tumors, or the blood-brain-barrier.    Examples of such amino acid sequences will be clear to the skilled    person. Some non-limiting examples are the small peptide vectors    (“Pep-trans vectors”) described in WO 03/026700 and in Temsamani et    al., Expert Opin. Biol. Ther., 1, 773 (2001); Temsamani and Vidal,    Drug Discov. Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp.    Ther., 296, 124-131 (2001), and the membrane translocator sequence    described by Zhao et al., Apoptosis, 8, 631-637 (2003). C-terminal    and N-terminal amino acid sequences for intracellular targeting of    antibody fragments are for example described by Cardinale et al.,    Methods, 34, 171 (2004). Other suitable techniques for intracellular    targeting involve the expression and/or use of so-called    “intrabodies” comprising a Nanobody of the invention, as mentioned    below;-   d) may form a “tag”, for example an amino acid sequence or residue    that allows or facilitates the purification of the Nanobody, for    example using affinity techniques directed against said sequence or    residue. Thereafter, said sequence or residue may be removed (e.g.    by chemical or enzymatical cleavage) to provide the Nanobody    sequence (for this purpose, the tag may optionally be linked to the    Nanobody sequence via a cleavable linker sequence or contain a    cleavable motif). Some preferred, but non-limiting examples of such    residues are multiple histidine residues, glutatione residues and a    myc-tag such as AAAEQKLISEEDLNGAA (SEQ ID NO: 476);-   e) may be one or more amino acid residues that have been    functionalized and/or that can serve as a site for attachment of    functional groups. Suitable amino acid residues and functional    groups will be clear to the skilled person and include, but are not    limited to, the amino acid residues and functional groups mentioned    herein for the derivatives of the Nanobodies of the invention.

According to another embodiment, a polypeptide of the inventioncomprises a Nanobody of the invention, which is fused at its aminoterminal end, at its carboxy terminal end, or both at its amino terminalend and at its carboxy terminal end to at least one further amino acidsequence, i.e. so as to provide a fusion protein comprising saidNanobody of the invention and the one or more further amino acidsequences. Such a fusion will also be referred to herein as a “Nanobodyfusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further amino acid sequences may ormay not change, alter or otherwise influence the (biological) propertiesof the Nanobody, and may or may not add further functionality to theNanobody or the polypeptide of the invention. Preferably, the furtheramino acid sequence is such that it confers one or more desiredproperties or functionalities to the Nanobody or the polypeptide of theinvention.

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005),

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the polypeptides of the invention,compared to the Nanobody of the invention per se. Some non-limitingexamples of such amino acid sequences are serum proteins, such as humanserum albumin (see for example WO 00/27435) or haptenic molecules (forexample haptens that are recognized by circulating antibodies, see forexample WO 98/22141).

The further amino acid sequence may also provide a second binding site,which binding site may be directed against any desired protein,polypeptide, antigen, antigenic determinant or epitope (including butnot limited to the same protein, polypeptide, antigen, antigenicdeterminant or epitope against which the Nanobody of the invention isdirected, or a different protein, polypeptide, antigen, antigenicdeterminant or epitope). For example, the further amino acid sequencemay provide a second binding site that is directed against a serumprotein (such as, for example, human serum albumin or another serumprotein such as IgG), so as to provide increased half-life in serum.Reference is for example made to EP 0 368 684, WO 91/01743, WO 01/45746and WO 04/003019 (in which various serum proteins are mentioned), theInternational application by applicant entitled “Nanobodies™ againstamyloid-beta and polypeptides comprising the same for the treatment ofdegenerative neural diseases such as Alzheimer's disease” (in whichvarious other proteins are mentioned), as well as to Harmsen et al.,Vaccine, 23 (41); 4926-42.

According to another embodiment, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a Nanobody of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain domain or to a natural orsynthetic analog of a V_(H) or V_(L) domain, again optionally via alinker sequence (including but not limited to other (single) domainantibodies, such as the dAb's described by Ward et al.).

The at least one Nanobody may also be linked to one or more (preferablyhuman) CH₁, CH₂ and/or CH₃ domains, optionally via a linker sequence.For instance, a Nanobody linked to a suitable CH₁ domain could forexample be used—together with suitable light chains—to generate antibodyfragments/structures analogous to conventional Fab fragments or F(ab′)2fragments, but in which one or (in case of an F(ab′)2 fragment) one orboth of the conventional V_(H) domains have been replaced by a Nanobodyof the invention. Also, two Nanobodies could be linked to a CH3 domain(optionally via a linker) to provide a construct with increasedhalf-life in vivo.

According to one specific embodiment of a polypeptide of the invention,one or more Nanobodies of the invention may linked to one or moreantibody parts, fragments or domains that confer one or more effectorfunctions to the polypeptide of the invention and/or may confer theability to bind to one or more Fc receptors. For example, for thispurpose, and without being limited thereto, the one or more furtheramino acid sequences may comprise one or more CH₂ and/or CH₃ domains ofan antibody, such as from a heavy chain antibody (as described herein)and more preferably from a conventional human 4-chain antibody; and/ormay form (part of) and Fc region, for example from IgG, from IgE or fromanother human Ig. For example, WO 94/04678 describes heavy chainantibodies comprising a Camelid V_(HH) domain or a humanized derivativethereof (i.e. a Nanobody), in which the Camelidae CH₂ and/or CH₃ domainhave been replaced by human CH₂ and CH₃ domains, so as to provide animmunoglobulin that consists of 2 heavy chains each comprising aNanobody and human CH2 and CH3 domains (but no CH1 domain), whichimmunoglobulin has the effector function provided by the CH2 and CH3domains and which immunoglobulin can function without the presence ofany light chains. Other amino acid sequences that can be suitably linkedto the Nanobodies of the invention so as to provide an effector functionwill be clear to the skilled person, and may be chosen on the basis ofthe desired effector function(s). Reference is for example made to WO04/058820, WO 99/42077 and WO 05/017148, as well as the review byHolliger and Hudson, supra. Coupling of a Nanobody of the invention toan Fc portion may also lead to an increased half-life, compared to thecorresponding Nanobody of the invention. For some applications, the useof an Fc portion and/or of constant domains (i.e. CH₂ and/or CH₃domains) that confer increased half-life without any biologicallysignificant effector function may also be suitable or even preferred.Other suitable constructs comprising one or more Nanobodies and one ormore constant domains with increased half-life in vivo will be clear tothe skilled person, and may for example comprise two Nanobodies linkedto a CH3 domain, optionally via a linker sequence. Generally, any fusionprotein or derivatives with increased half-life will preferably have amolecular weight of more than 50 kD, the cut-off value for renalabsorption.

The further amino acid sequences may also form a signal sequence orleader sequence that directs secretion of the Nanobody or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro-form of the polypeptide of theinvention, depending on the host cell used to express the polypeptide ofthe invention).

The further amino acid sequence may also form a sequence or signal thatallows the Nanobody or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows theNanobody or polypeptide of the invention to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Suitable examples of such amino acid sequences willbe clear to the skilled person, and for example include, but are notlimited to, the “Peptrans” vectors mentioned above, the sequencesdescribed by Cardinale et al. and the amino acid sequences and antibodyfragments known per se that can be used to express or produce theNanobodies and polypeptides of the invention as so-called “intrabodies”,for example as described in WO 94/02610, WO 95/22618, U.S. Pat. No.7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and inCattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Developmentand Applications. Landes and Springer-Verlag; and in Kontermann, Methods34, (2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a(cyto)toxic protein or polypeptide. Examples of such toxic proteins andpolypeptides which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic polypeptide of the invention will beclear to the skilled person and can for example be found in the priorart cited above and/or in the further description herein. One example isthe so-called ADEPT™ technology WO 03/055527.

According to one non-limiting embodiment, one or more amino acidresidues can be added to, inserted in and/or substituted in the aminoacid sequence of a Nanobody or polypeptide of the invention, so as toprovide one or more specific amino acid residues for attachment of aPEG-group.

The efficacy of protein pharmaceuticals depends on its potency toneutralize the target but also on the intrinsic pharmacokinetics of thepotential drug. Because the kidney generally filters out molecules below60,000 Daltons (Da), efforts to reduce clearance have focused onincreasing the molecular weight of the biopharmaceutical through proteinfusions (Syed et al., 1997), glycosylations or modification withpolyethylene glycol polymers, i.e., PEGylation (Lee et al., 1999;Abuchowski et al., 1977; Nucci et al., 1991; Lecolley, et al. ChemCommun, 2004; Tao et al., J Am Chem Soc, 2004; Mantovani et al., 2005).These methods successfully extend the in vivo exposure of thebiopharmaceutical.

Alternatively, the half-life can be extended using another pegylatingagent, POLY PEG for conjugation to the bivalent Nanobodies, TNF56 orTNF55. POLY PEG are comb shape polymers with PEG teeth on a methacrylicbackbone. POLY PEGs can vary on the length of the PEG chain, on themethacrylic backbone and on the active end-group which determines themethod of conjugation of the POLY PEG to the Nanobody. Site-specificconjugation to the C-terminal cysteine present in the Nanobodies can beachieved through the active maleimide end-group in the POLY PEG.

The invention also encompasses any Nanobody of the invention and/orpolypeptide of the invention that has been glycosylated at one or moreamino acid positions, usually depending upon the host used to expressthe Nanobody or polypeptide of the invention (as further describedbelow).

According to one non-limiting embodiment, one or more amino acidresidues can be added to, inserted in and/or substituted in the aminoacid sequence of a Nanobody or polypeptide of the invention, so as toprovide one or more specific amino acid residues and/or a site that canbe glycosylated by the host organism used. By means of a preferred, butnon-limiting example, the N-residue on position 50 within CDR2 of aNanobody of the invention can for example be replaced by a Q, D or Sresidue so as to provide a glycosylation site, e.g. for glycosylation byPichia.

According to another embodiment, a polypeptide of the invention cancomprise the amino acid sequence of a Nanobody, which is fused at itsamino terminal end, at its carboxy terminal end, or both at its aminoterminal end and at its carboxy terminal end with at least one furtheramino acid sequence.

Again, said further amino acid sequence(s) may or may not change, alteror otherwise influence the (biological) properties of the Nanobody andmay or may not add further functionality to the Nanobody.

For example, according to one preferred, but non-limiting embodiment,said further amino acid sequence may comprise at least one furtherNanobody, so as to provide a polypeptide of the invention that comprisesat least two, such as three, four or five, Nanobodies, in which saidNanobodies may optionally be linked via one or more linker sequences (asdefined herein).

Polypeptides of the invention comprising two or more Nanobodies willalso referred to herein as “multivalent” polypeptides. For example a“bivalent” polypeptide of the Invention comprises two Nanobodies,optionally linked via a linker sequence, whereas a “trivalent”polypeptide of the invention comprises three Nanobodies, optionallylinked via two linker sequences; etc.

In a multivalent polypeptide of the invention, the two or moreNanobodies may be the same or different. For example, the two or moreNanobodies in a multivalent polypeptide of the invention:

-   -   may be directed against the same antigen, i.e. against the same        parts or epitopes of said antigen or against two or more        different parts or epitopes of said antigen; and/or:    -   may be directed against the different antigens;        or a combination thereof.

Thus, a bivalent polypeptide of the invention for example:

-   -   may comprise two identical Nanobodies;    -   may comprise a first Nanobody directed against a first part or        epitope of an antigen and a second Nanobody directed against the        same part or epitope of said antigen or against another part or        epitope of said antigen;    -   or may comprise a first Nanobody directed against a first        antigen and a second

Nanobody directed against a second antigen different from said firstantigen; whereas a trivalent Polypeptide of the Invention for example:

-   -   may comprises three identical or different Nanobodies directed        against the same or different parts or epitopes of the same        antigen;    -   may comprise two identical or different Nanobodies directed        against the same or different parts or epitopes on a first        antigen and a third Nanobody directed against a second antigen        different from said first antigen; or    -   may comprise a first Nanobody directed against a first antigen,        a second Nanobody directed against a second antigen different        from said first antigen, and a third Nanobody directed against a        third antigen different from said first and second antigen,

Polypeptides of the invention that contain at least two Nanobodies, inwhich at least one Nanobody is directed against a first antigen and atleast one Nanobody is directed against a second antigen different fromthe first antigen, will also be referred to as “multispecific”Nanobodies. Thus, a “bispecific” Nanobody is a Nanobody that comprisesat least one Nanobody directed against a first antigen and at least onefurther Nanobody directed against a second antigen, whereas a“trispecific” Nanobody is a Nanobody that comprises at least oneNanobody directed against a first antigen, at least one further Nanobodydirected against a second antigen, and at least one further Nanobodydirected against a third antigen; etc.

Accordingly, in their simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against a first antigenand a second Nanobody directed against a second antigen, in which saidfirst and second Nanobody may optionally be linked via a linker sequence(as defined herein); whereas a trispecific polypeptide of the inventionin its simplest form is a trivalent polypeptide of the invention (asdefined herein), comprising a first Nanobody directed against a firstantigen, a second Nanobody directed against a second antigen and a thirdNanobody directed against a third antigen, in which said first, secondand third Nanobody may optionally be linked via one or more, and inparticular one and more in particular two, linker sequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise any number of Nanobodiesdirected against two or more different antigens.

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, as well as to EP 0 822985.

In the polypeptides of the invention, the one or more Nanobodies and theone or more polypeptides may be directly linked to each other (as forexample described in WO 99/23221) and/or may be linked to each other viaone or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each Nanobody by itselfforms a complete antigen-binding site).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, poly(ethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the Nanobodies of theinvention). For example, linkers containing one or more charged aminoacid residues (see Table A-3 above) can provide improved hydrophilicproperties, whereas linkers that form or contain small epitopes or tagscan be used for the purposes of detection, identification and/orpurification. Again, based on the disclosure herein, the skilled personwill be able to determine the optimal linkers for use in a specificpolypeptide of the invention, optionally after on some limited routineexperiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after on some limited routine experiments.

Linkers for use in multivalent and multispecific polypeptides will beclear to the skilled person, and for example include gly-ser linkers,for example of the type (gly_(x)ser_(y))_(z), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077, hinge likeregions such as the hinge regions of naturally occurring heavy chainantibodies or similar sequences. For other suitable linkers, referenceis also made to the general background art cited above. Someparticularly preferred linkers are given in SEQ ID NO's 68 and 69.

Linkers can also provide some functionality for the multivalent ormultispecific polypeptide. For example, linkers containing one or morecharged amino acid residues (see Table 1 above) can provide improvedhydrophilic properties, whereas linkers that form or contain smallepitopes or tags can be used for the purposes of detection,identification and/or purification.

As mentioned herein, in a protein or polypeptide of the invention, theanti-TNF Nanobodies mentioned herein are preferably linked in such a waythat said protein or polypeptide, upon binding to a TNF trimer, iscapable inhibiting or reducing the TNF receptor crosslinking that ismediated by said TNF trimer and/or the signal transduction that ismediated by such receptor crosslinking; and/or in such a way that theprotein or polypeptide is capable of intramolecular binding to at leasttwo TNF receptor binding sites on a TNF trimer. Suitable linkers are asdescribed herein.

As also mentioned herein, whether a protein or polypeptide providesintermolecular binding or extramolecular binding can be assessed (atleast initially) by a size exclusion chromatography. By Size ExclusionChromatography the complexes of TNF-alpha and antibodies can be analyzedfor determining the number and the ratio of antibody and TNF-alphamolecules in the complex. From these data it can be deduced if inter- orintramolecular binding occurs, as was done by Santora and colleagues(Santora, L. C., et al, Anal Biochem. 2001) for establishing thestoichiometry of binding of monoclonal antibody D2E7 (Humira) toTNF-alpha□ at different ratios of antibody and target. From themolecular weight of the complex it was concluded that three antibodymolecules complexed with three TNF trimers, thereby indicating that theantibody binds in an intermolecular mode. Similar experiments wereperformed with bivalent Nanobodies, in which a very short linker inducedthe formation of large molecular complexes, which were obtained byintermolecular bonds. However, the same bivalent Nanobodies constructswith longer linkers eluted from the gel filtration column as discretesmall complexes, thereby demonstrating that intramolecular bonds wereformed. Combined with the bioassay data, in which the longer linkercontaining Nanobody TNF1 had an optimal potency (complete neutralizationof amount of TNF used in the assay, i.e. 10 pM), it can be concludedthat intramolecular binding of the bivalent Nanobody efficientlyprevents cross-linking of two cell bound receptors and the associatedreceptor activation. Known monoclonal antibodies such as Humira orRemicade can not form such intramolecular bonds, leaving always tworeceptor bindingsites on the trimeric TNF molecule to a certain degreeavailable for interaction with cell bound receptor, which translatesinto less potent neutralization as measured in the bioassay.

Alternatively, whether a protein or polypeptide provides intermolecularbinding or extramolecular binding can be assessed by crystallographyand/or molecular modelling (or other suitable in silico techniques). Amodel of a trimeric TNF30/TNF-alpha complex was generated based on thecrystal structure of the monomeric wild type TNF1/TNF-alpha complex.From this structure the final TNF30-linker-ALB8-linker-TNF30 constructwas modeled. The TNF30-linker-ALB8-linker-TNF30 construct was modelledstarting from the trimer of TNFα with two TNF30 molecules bound. As thestructure of the ALB8 is not known, a third TNF30 molecule was usedinstead, which was placed in between the other two Nanobodies along theline between the N- and C-termini. The 9 amino acid linkers were thenadded manually.

The model is shown in FIG. 62. Clearly, the 9 amino acid linkerstogether with the ALB8 provide ample room to span the about 66 Å betweenthe two TNF30 domains bound to TNFα. ALB8 by itself already spans 40 Å,and each linker can span another ˜27 Å in completely extendedconformation. As a result, the ALB8 has quite some flexibility ofmovement, and it is not expected that its binding to albumin wouldinterfere much with the binding to TNFα.

Moreover, it is likely that the linkers can be shortened withoutaffecting avidity, especially in the case of the linker that isC-terminal of ALB8. This may have the beneficial effect of increasedbinding to the same TNFα trimer versus crosslinking trimers, because theprobability that the second TNF30 associates with a different TNFαincreases with the length of the linker.

As also further described herein, a multispecific polypeptide of theinvention directed against a desired antigen and against at least oneserum protein, such as the serum proteins mentioned hereinbelow, and inparticular against human serum albumin, may show increased half-life inserum, compared to the corresponding monovalent Nanobody.

As mentioned hereinabove, the methods described herein are particularlysuited for generating such multivalent of multispecific polypeptides ofthe invention.

In a polypeptide of the invention, the at least one Nanobody may also belinked to a conventional V_(H) domain or to a natural or syntheticanalog of a V_(H) domain, optionally via a linker sequence.

In a polypeptide of the invention, the at least one Nanobody may also belinked to a V_(L) domain or to a natural or synthetic analog of a V_(L)domain, optionally via a linker sequence, so as to provide a polypeptideof the invention that is in the form analogous to a conventional scFvfragment, but containing a Nanobody instead of a V_(H) domain.

In a polypeptide of the invention, the at least one Nanobody may also belinked to one or more of a CH1, CH2 and/or CH3 domain, optionally via alinker sequence. For instance, a Nanobody linked to a suitable CH1domain could for example be used—together with suitable light chains—togenerate antibody fragments/structures analogous to conventional Fabfragments or F(ab′)₂ fragments, but in which one or (in case of anF(ab′)₂ fragment) one or both of the conventional V_(H) domains havebeen replaced by a Nanobody. Such fragments may also be heterospecificor bispecific, i.e. directed against two or more antigens. A Nanobodylinked to suitable CH2 and CH3 domains, for example derived fromCamelids, could be used to form a monospecific or bispecific heavy chainantibody. Finally, a Nanobody linked to suitable CH1, CH2 and CH3domains, for example derived from a human being, could be used—togetherwith suitable light chains—to form an antibody that is analogous to aconventional 4-chain antibody, but in which one or both of theconventional V_(H) domains have been replaced by a Nanobody.

Also, in addition to the one or more Nanobodies, Polypeptides of theInvention can also contain functional groups, moieties or residues, forexample therapeutically active substances, such as those mentionedbelow, and/or markers or labels, such as fluorescent markers, isotopes,etc., as further described hereinbelow.

The Nanobodies of the invention, the polypeptides of the invention, andnucleic acids encoding the same, can be prepared in a manner known perse, as will be clear to the skilled person from the further descriptionherein. Some preferred, but non-limiting methods for preparing theNanobodies, polypeptides and nucleic acids include the methods andtechniques mentioned above and/or further described hereinbelow.

As will be clear to the skilled person, one particularly useful methodfor preparing a Nanobody and/or a polypeptide of the invention generallycomprises the steps of:

-   -   the expression, in a suitable host cell or host organism (also        referred to herein as a “host of the invention”) or in another        suitable expression system of a nucleic acid that encodes said        Nanobody or polypeptide of the invention (also referred to        herein as a “nucleic acid of the invention”), optionally        followed by:    -   isolating and/or purifying the Nanobody or polypeptide of the        invention thus obtained.

In particular, such a method may comprise the steps of:

-   -   cultivating and/or maintaining a host of the invention under        conditions that are such that said host of the invention        expresses and/or produces at least one Nanobody and/or        polypeptide of the invention; optionally followed by:    -   isolating and/or purifying the Nanobody or polypeptide of the        invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one embodiment of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined hereinabove.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can forexample be subjected to site-directed mutagenesis, so as to provide anucleic acid of the invention encoding said analog. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding a Nanobody and for example nucleic acids encoding oneor more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring GPCR as a template. These and other techniques willbe clear to the skilled person, and reference is again made to thestandard handbooks, such as Sambrook et al. and Ausubel et al.,mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to hereinbelow. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable independentreplication, maintenance and/or inheritance in the intended hostorganism. For instance, the genetic constructs of the invention may bein the form of a vector, such as for example a plasmid, cosmid, YAC, aviral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting embodiment, a genetic construct of theinvention comprises

-   a) at least one nucleic acid of the invention; operably connected to-   b) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;-   c) and optionally also-   d) one or more further elements of genetic constructs known per se;    in which the terms “regulatory element”, “promoter”, “terminator”    and “operably connected” have their usual meaning in the art (as    further described below); and in which said “further elements”    present in the genetic constructs may for example be 3′- or 5′-UTR    sequences, leader sequences, selection markers, expression    markers/reporter genes, and/or elements that may facilitate or    increase (the efficiency of) transformation or integration. These    and other suitable elements for such genetic constructs will be    clear to the skilled person, and may for instance depend upon the    type of construct used, the intended host cell or host organism; the    manner in which the nucleotide sequences of the invention of    interest are to be expressed (e.g. via constitutive, transient or    inducible expression); and/or the transformation technique to be    used.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of’ said promotor). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

Some particularly preferred promoters include, but are not limited to,promoters known per se for the expression in bacterial cells, such asthose mentioned hereinbelow and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriateselection conditions—host cells and/or host organisms that have been(succesfully) transformed with the nucleotide sequence of the inventionto be distinguished from host cells/organisms that have not been(succesfully) transformed. Some preferred, but non-limiting examples ofsuch markers are genes that provide resistance against antibiotics (suchas kanamycine or ampicilline), genes that provide for temperatureresistance, or genes that allow the host cell or host organism to bemaintained in the absence of certain factors, compounds and/or (food)components in the medium that are essential for survival of thenon-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or hostorganism—it allows for the desired post-translational modificationsand/or such that it directs the transcribed mRNA to a desired part ororganelle of a cell. A leader sequence may also allow for secretion ofthe expression product from said cell. As such, the leader sequence maybe any pro-, pre-, or prepro-sequence operable in the host cell or hostorganism. Leader sequences may not be required for expression in abacterial cell.

An expression marker or reporter gene should be such that—in the hostcell or host organism—it allows for detection of the expression of (agene or nucleotide sequence present on) the genetic construct. Anexpression marker may optionally also allow for the localisation of theexpressed product, e.g. in a specific part or organelle of a cell and/orin (a) specific cell(s), tissue(s), organ(s) or part(s) of amulticellular organism. Such reporter genes may also be expressed as aprotein fusion with the amino acid sequence of the invention. Somepreferred, but non-limiting examples include fluorescent proteins suchas GFP.

Some preferred, but non-limiting examples of suitable promoters,terminator and further elements include those used in the Examplesbelow. For some (further) non-limiting examples of the promoters,selection markers, leader sequences, expression markers and furtherelements that may be present/used in the genetic constructs of theinvention—such as terminators, transcriptional and/or translationalenhancers and/or integration factors—reference is made to the generalhandbooks such as Sambrook et al. and Ausubel et al. mentioned above, aswell as to the examples that are given in WO 95/07463, WO 96/23810, WO95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO98/21355, U.S. Pat. Nos. 6,207,410, 5,693,492 and EP 1 085 089. Otherexamples will be clear to the skilled person. Reference is also made tothe general background art cited above and the further references citedhereinbelow.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned below.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism. Thehost cell or host organism may be any suitable (fungal, prokaryotic oreukaryotic) cell or cell line or any suitable fungal, prokaryotic oreukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        lividans; of Staphylococcus, for example of Staphylococcus        carnosus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosaccharomyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorpha;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for Example of Arxula Adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example derived a cell or        cell line derived from a human, from the mammals including but        not limited to-cells, BHK-cells (for example BHK-21 cells) and        human cells or cell lines such as HeLa, COS (for example COS-7)        and PER.C6 cells;        as well as all other hosts or host cells known per se for the        expression and production of antibodies and antibody fragments        (including but not limited to (single) domain antibodies and        ScFv fragments), which will be clear to the skilled person.        Reference is also made to the general background art cited        hereinabove, as well as to for example WO 94/29457; WO 96/34103;        WO 99/42077; Frenken et al., (1998), supra; Riechmann and        Muyldermans, (1999), supra; van der Linden, (2000), supra;        Thomassen et al., (2002), supra; Joosten et al., (2003), supra;        Joosten et al., (2005), supra; and the further references cited        herein.

The Nanobodies and polypeptides of the invention can also be introducedand expressed in one or more cells, tissues or organs of a multicellularorganism, for example for prophylactic and/or therapeutic purposes (e.g.as a gene therapy). For this purpose, the nucleotide sequences of theinvention may be introduced into the cells or tissues in any suitableway, for example as such (e.g. using liposomes) or after they have beeninserted into a suitable gene therapy vector (for example derived fromretroviruses such as adenovirus, or parvoviruses such asadeno-associated virus). As will also be clear to the skilled person,such gene therapy may be performed in vivo and/or in situ in the body ofa patient by administering a nucleic acid of the invention or a suitablegene therapy vector encoding the same to the patient or to specificcells or a specific tissue or organ of the patient; or suitable cells(often taken from the body of the patient to be treated, such asexplanted lymphocytes, bone marrow aspirates or tissue biopsies) may betreated in vitro with a nucleotide sequence of the invention and then besuitably (re-)introduced into the body of the patient. All this can beperformed using gene therapy vectors, techniques and delivery systemswhich are well known to the skilled person, for example, Culver, K. W.,“Gene Therapy”, 1994, p. xii, Mary Ann Liebert, Inc., Publishers, NewYork, N.Y.; Giordano, Nature F Medicine 2 (1996), 534-539; Schaper,Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813;Verma, Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374;Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998),30-36; Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.:811 (1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51;Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S.Pat. Nos. 5,580,859; 5,5895,466; or Schaper, Current Opinion inBiotechnology 7 (1996), 635-640. For example, in situ expression of ScFvfragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and ofdiabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has beendescribed in the art.

For expression of the Nanobodies in a cell, they may also be expressedas so-called “intrabodies”, as for example described in WO 94/02610, WO95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. &Biocca, S. (1997) Intracellular Antibodies: Development andApplications. Landes and Springer-Verlag; and in Kontermann, Methods 34,(2004), 163-170.

For production, the Nanobodies and polypeptides of the invention can forexample also be produced in the milk of transgenic mammals, for examplein the milk of rabbits, cows, goats or sheep (see for example U.S. Pat.Nos. 6,741,957, 6,304,489 and 6,849,992 for general techniques forintroducing transgenes into mammals), in plants or parts of plantsincluding but not limited to their leaves, flowers, fruits, seed, rootsor tubers (for example in tobacco, maize, soybean or alfalfa) or in forexample pupae of the silkworm Bombyx mori.

Furthermore, the Nanobodies and polypeptides of the invention can alsobe expressed and/or produced in cell-free expression systems, andsuitable examples of such systems will be clear to the skilled person.Some preferred, but non-limiting examples include expression in thewheat germ system; in rabbit reticulocyte lysates; or in the E. coliZubay system.

As mentioned above, one of the advantages of the use of Nanobodies isthat the polypeptides based thereon can be prepared through expressionin a suitable bacterial system, and suitable bacterial expressionsystems, vectors, host cells, regulatory elements, etc., will be clearto the skilled person, for example from the references cited above. Itshould however be noted that the invention in its broadest sense is notlimited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,Polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of Nanobodies or Nanobody-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceuticalexpression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a Nanobody-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast are usually leads to a glycosylation pattern that differsfrom human glycosylation. Nevertheless, it should be understood that allthe foregoing host cells and expression systems can be used in theinvention, depending on the desired Nanobody or protein to be obtained.

Thus, according to one non-limiting embodiment of the invention, theNanobody or polypeptide of the invention is glycosylated. According toanother non-limiting embodiment of the invention, the Nanobody orpolypeptide of the invention is non-glycosylated.

According to one preferred, but non-limiting embodiment of theinvention, the Nanobody or polypeptide of the invention is produced in abacterial cell, in particular a bacterial cell suitable for large scalepharmaceutical production, such as cells of the strains mentioned above.

According to another preferred, but non-limiting embodiment of theinvention, the Nanobody or polypeptide of the invention is produced in ayeast cell, in particular a yeast cell suitable for large scalepharmaceutical production, such as cells of the species mentioned above.

According to yet another preferred, but non-limiting embodiment of theinvention, the Nanobody or polypeptide of the invention is produced in amammalian cell, in particular in a human cell or in a cell of a humancell line, and more in particular in a human cell or in a cell of ahuman cell line that is suitable for large scale pharmaceuticalproduction, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the Nanobodies and theproteins of the invention, the Nanobodies and proteins of the inventioncan be produced either intracellullarly (e.g. in the cytosol, in theperiplasma or in inclusion bodies) and then isolated from the host cellsand optionally further purified; or can be produced extracellularly(e.g. in the medium in which the host cells are cultured) and thenisolated from the culture medium and optionally further purified. Wheneukaryotic hosts cells are used, extracellular production is usuallypreferred since this considerably facilitates the further isolation anddownstream processing of the Nanobodies and proteins obtained. Bacterialcells such as the strains of E. coli mentioned above normally do notsecrete proteins extracellularly, except for a few classes of proteinssuch as toxins and hemolysin, and secretory production in E. coli refersto the translocation of proteins across the inner membrane to theperiplasmic space. Periplasmic production provides several advantagesover cytosolic production. For example, the N-terminal amino acidsequence of the secreted product can be identical to the natural geneproduct after cleavage of the secretion signal sequence by a specificsignal peptidase. Also, there appears to be much less protease activityin the periplasm than in the cytoplasm. In addition, proteinpurification is simpler due to fewer contaminating proteins in theperiplasm. Another advantage is that correct disulfide bonds may formbecause the periplasm provides a more oxidative environment than thecytoplasm. Proteins overexpressed in E. coli are often found ininsoluble aggregates, so-called inclusion bodies. These inclusion bodiesmay be located in the cytosol or in the periplasm; the recovery ofbiologically active proteins from these inclusion bodies requires adenaturation/refolding process. Many recombinant proteins, includingtherapeutic proteins, are recovered from inclusion bodies.Alternatively, as will be clear to the skilled person, recombinantstrains of bacteria that have been genetically modified so as to secretea desired protein, and in particular a Nanobody or a polypeptide of theinvention, can be used.

Thus, according to one non-limiting embodiment of the invention, theNanobody or polypeptide of the invention is a Nanobody or polypeptidethat has been produced intracellularly and that has been isolated fromthe host cell, and in particular from a bacterial cell or from aninclusion body in a bacterial cell. According to another non-limitingembodiment of the invention, the Nanobody or polypeptide of theinvention is a Nanobody or polypeptide that has been producedextracellularly, and that has been isolated from the medium in which thehost cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude,

-   -   for expression in E. coli: lac promoter (and derivatives thereof        such as the lacUV5 promoter); arabinose promoter; left- (PL) and        rightward (PR) promoter of phage lambda; promoter of the trp        operon; hybrid lac/trp promoters (tac and trc); T7-promoter        (more specifically that of T7-phage gene 10) and other T-phage        promoters; promoter of the Tn10 tetracycline resistance gene;        engineered variants of the above promoters that include one or        more copies of an extraneous regulatory operator sequence;    -   for expression in S. cerevisiae: constitutive: ADH1 (alcohol        dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),        GAPDH (glyceraldehydes-3-phosphate dehydrogenase); PGK1        (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:        GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol        dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper        metallothionein); heterologous: CaMV (cauliflower mosaic virus        35S promoter);    -   for expression in Pichia pastoris: the AOX1 promoter (alcohol        oxidase I)    -   for expression in mammalian cells: human cytomegalovirus (hCMV)        immediate early enhancer/promoter; human cytomegalovirus (hCMV)        immediate early promoter variant that contains two tetracycline        operator sequences such that the promoter can be regulated by        the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)        promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)        enhancer/promoter; elongation factor 1α (hEF-1α) promoter from        human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1        long terminal repeat promoter; β-actin promoter;        Some preferred, but non-limiting vectors for use with these host        cells include:    -   vectors for expression in mammalian cells: pMAMneo (Clontech),        pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene),        EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),        pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo        (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and        1ZD35 (ATCC 37565), as well as viral-based expression systems,        such as those based on adenovirus;    -   vectors for expression in bacterials cells: pET vectors        (Novagen) and pQE vectors (Qiagen);    -   vectors for expression in yeast or other fungal cells: pYES2        (Invitrogen) and Pichia expression vectors (Invitrogen);    -   vectors for expression in insect cells: pBlueBacII (Invitrogen)        and other baculovirus vectors    -   vectors for expression in plants or plant cells: for example        vectors based on cauliflower mosaic virus or tobacco mosaic        virus, suitable strains of Agrobacterium, or Ti-plasmid based        vectors.        Some preferred, but non-limiting secretory sequences for use        with these host cells include:    -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,        OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the        like; TAT signal peptide, hemolysin C-terminal secretion signal        for use in yeast: α-mating factor prepro-sequence, phosphatase        (pho1), invertase (Suc), etc.,    -   for use in mammalian cells: indigenous signal in case the target        protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal        peptide; etc.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form of a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence of the invention (and in case of ahost organism: in at least one cell, part, tissue or organ thereof). Theinvention also includes further generations, progeny and/or offspring ofthe host cell or host organism of the invention, that may for instancebe obtained by cell division or by sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence of the invention isexpressed/produced. Suitable conditions will be clear to the skilledperson and will usually depend upon the host cell/host organism used, aswell as on the regulatory elements that control the expression of the(relevant) nucleotide sequence of the invention. Again, reference ismade to the handbooks and patent applications mentioned above in theparagraphs on the genetic constructs of the invention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acid sequenceof the invention may (first) be generated in an immature form (asmentioned above), which may then be subjected to post-translationalmodification, depending on the host cell/host organism used. Also, theamino acid sequence of the invention may be glycosylated, againdepending on the host cell/host organism used.

The amino acid sequence of the invention may then be isolated from thehost cell/host organism and/or from the medium in which said host cellor host organism was cultivated, using protein isolation and/orpurification techniques known per se, such as (preparative)chromatography and/or electrophoresis techniques, differentialprecipitation techniques, affinity techniques (e.g. using a specific,cleavable amino acid sequence fused with the amino acid sequence of theinvention) and/or preparative immunological techniques (i.e. usingantibodies against the amino acid sequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention ofthe inventions may be formulated as a pharmaceutical preparationcomprising at least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described hereinbelow.

Generally, the Nanobodies and polypeptides of the invention can beformulated and administered in any suitable manner known per se, forwhich reference is for example made to the general background art citedabove (and in particular to WO 04/041862, WO 04/041863, WO 04/041865 andWO 04/041867) as well as to the standard handbooks, such as Remington'sPharmaceutical Sciences, 18^(th) Ed., Mack Publishing Company, USA(1990) or Remington, the Science and Practice of Pharmacy, 21th Edition,Lippincott Williams and Wilkins (2005).

For example, the Nanobodies and polypeptides of the invention may beformulated and administered in any manner known per se for conventionalantibodies and antibody fragments (including ScFv's and diabodies) andother pharmaceutically active proteins. Such formulations and methodsfor preparing the same will be clear to the skilled person, and forexample include preparations suitable for parenteral administration (forexample intravenous, intraperitoneal, subcutaneous, intramuscular,intraluminal, intra-arterial or intrathecal administration) or fortopical (i.e. transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andaqueous buffers and solutions such as physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution;water oils; glycerol; ethanol; glycols such as propylene glycol or aswell as mineral oils, animal oils and vegetable oils, for example peanutoil, soybean oil, as well as suitable mixtures thereof. Usually, aqueoussolutions or suspensions will be preferred.

The Nanobodies and polypeptides of the invention can also beadministered using gene therapy methods of delivery. See, e.g., U.S.Pat. No. 5,399,346, which is incorporated by reference in its entirety.Using a gene therapy method of delivery, primary cells transfected withthe gene encoding a Nanobody or polypeptide of the invention canadditionally be transfected with tissue specific promoters to targetspecific organs, tissue, grafts, tumors, or cells and can additionallybe transfected with signal and stabilization sequences for subcellularlylocalized expression.

Thus, the Nanobodies and polypeptides of the invention may besystemically administered, e.g., orally, in combination with apharmaceutically acceptable vehicle such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the Nanobodies and polypeptides of the invention may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of the Nanobody or polypeptide of the invention.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the Nanobody orpolypeptide of the invention in such therapeutically useful compositionsis such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the Nanobodies and polypeptides of the invention, sucrose orfructose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, the Nanobodies and polypeptides of the inventionmay be incorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The Nanobodies and polypeptides of the invention may also beadministered intravenously or intraperitoneally by infusion orinjection. Solutions of the Nanobodies and polypeptides of the inventionor their salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating theNanobodies and polypeptides of the invention in the required amount inthe appropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filter sterilization. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze dryingtechniques, which yield a powder of the active ingredient plus anyadditional desired ingredient present in the previously sterile-filteredsolutions.

For topical administration, the Nanobodies and polypeptides of theinvention may be applied in pure form, i.e., when they are liquids.However, it will generally be desirable to administer them to the skinas compositions or formulations, in combination with a dermatologicallyacceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the Nanobodies and polypeptides of the invention can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the Nanobodies and polypeptides of the invention to the skin areknown to the art; for example, see Jacquet et al. (U.S. Pat. No.4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No.4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the Nanobodies and polypeptides of the invention canbe determined by comparing their in vitro activity, and in vivo activityin animal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949.

Generally, the concentration of the Nanobodies and polypeptides of theinvention in a liquid composition, such as a lotion, will be from about0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in asemi-solid or solid composition such as a gel or a powder will be about0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the Nanobodies and polypeptides of the invention requiredfor use in treatment will vary not only with the particular Nanobody orpolypeptide selected but also with the route of administration, thenature of the condition being treated and the age and condition of thepatient and will be ultimately at the discretion of the attendantphysician or clinician. Also the dosage of the Nanobodies andpolypeptides of the invention varies depending on the target cell,tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one TNF-relates disease or disorder asmentioned herein, said method comprising administering, to a subject inneed thereof, a pharmaceutically active amount of a Nanobody of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk from, the diseases anddisorders mentioned herein.

The invention also relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering a Nanobody or polypeptide of theinvention to a patient, said method comprising administering, to asubject in need thereof, a pharmaceutically active amount of a Nanobodyof the invention, of a polypeptide of the invention, and/or of apharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of a Nanobody of the invention, of apolypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In another embodiment, the invention relates to a method forimmunotherapy, and in particular for passive immunotherapy, which methodcomprises administering, to a subject suffering from or at risk of thediseases and disorders mentioned herein, a pharmaceutically activeamount of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In the above methods, the Nanobodies and/or polypeptides of theinvention and/or the compositions comprising the same can beadministered in any suitable manner, depending on the specificpharmaceutical formulation or composition to be used. Thus, theNanobodies and/or polypeptides of the invention and/or the compositionscomprising the same can for example be administered orally,intraperitoneally (e.g. intravenously, subcutaneously, intramuscularly,or via any other route of administration that circumvents thegastrointestinal tract), intranasally, transdermally, topically, bymeans of a suppository, by inhalation, again depending on the specificpharmaceutical formulation or composition to be used. The clinician willbe able to select a suitable route of administration and a suitablepharmaceutical formulation or composition to be used in suchadministration, depending on the disease or disorder to be prevented ortreated and other factors well known to the clinician.

The Nanobodies and/or polypeptides of the invention and/or thecompositions comprising the same are administered according to a regimeof treatment that is suitable for preventing and/or treating the diseaseor disorder to be prevented or treated. The clinician will generally beable to determine a suitable treatment regimen, depending on factorssuch as the disease or disorder to be prevented or treated, the severityof the disease to be treated and/or the severity of the symptomsthereof, the specific Nanobody or polypeptide of the invention to beused, the specific route of administration and pharmaceuticalformulation or composition to be used, the age, gender, weight, diet,general condition of the patient, and similar factors well known to theclinician.

Generally, the treatment regimen will comprise the administration of oneor more Nanobodies and/or polypeptides of the invention, or of one ormore compositions comprising the same, in one or more pharmaceuticallyeffective amounts or doses. The specific amount(s) or doses toadministered can be determined by the clinician, again based on thefactors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific Nanobody andpolypeptide of the invention to be used, the specific route ofadministration and the specific pharmaceutical formulation orcomposition used, the Nanobodies and polypeptides of the invention willgenerally be administered in an amount between 1 gram and 0.01 microgramper kg body weight per day, preferably between 0.1 gram and 0.1microgram per kg body weight per day, such as about 1, 10, 100 or 1000microgram per kg body weight per day, either continuously (e.g. byinfusion), as a single daily dose or as multiple divided doses duringthe day. The clinician will generally be able to determine a suitabledaily dose, depending on the factors mentioned herein. It will also beclear that in specific cases, the clinician may choose to deviate fromthese amounts, for example on the basis of the factors cited above andhis expert judgment. Generally, some guidance on the amounts to beadministered can be obtained from the amounts usually administered forcomparable conventional antibodies or antibody fragments against thesame target administered via essentially the same route, taking intoaccount however differences in affinity/avidity, efficacy,biodistribution, half-life and similar factors well known to the skilledperson.

Usually, in the above method, a single Nanobody or polypeptide of theinvention will be used. It is however within the scope of the inventionto use two or more Nanobodies and/or polypeptides of the invention incombination.

The Nanobodies and polypeptides of the invention may also be used incombination with one or more further pharmaceutically active compoundsor principles, i.e. as a combined treatment regimen, which may or maynot lead to a synergistic effect. Again, the clinician will be able toselect such further compounds or principles, as well as a suitablecombined treatment regimen, based on the factors cited above and hisexpert judgement.

In particular, the Nanobodies and polypeptides of the invention may beused in combination with other pharmaceutically active compounds orprinciples that are or can be used for the prevention and/or treatmentof the diseases and disorders cited herein, as a result of which asynergistic effect may or may not be obtained. Examples of suchcompounds and principles, as well as routes, methods and pharmaceuticalformulations or compositions for administering them will be clear to theclinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are administered to be simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one Nanobody of the invention or atleast one polypeptide of the invention and at least one suitable carrier(i.e. a carrier suitable for veterinary use), and optionally one or morefurther active substances.

The invention also relates to the use of a Nanobody of the inventionand/or of a polypeptide of the invention in the preparation of apharmaceutical composition, in particular in the preparation of apharmaceutical composition for the prevention and/or treatment(including but not limiting to the alleviation of at least one symptom)of a disease or disorder mediated by TNF-alpha and/or associated withTNF-alpha (for example associated with an abnormal activity ofTNF-alpha, abnormal levels of TNF-alpha, abnormal expression ofTNF-alpha and/or abnormal sensitivity or response to TNF-alpha), or ofone of the biological phenomena associated with TNF-alpha, such as oneof the diseases or disorders mentioned above.

The invention also relates to a method for preventing and/or treating(including but not limiting to the alleviation of at least one symptom)of a disease or disorder mediated by TNF-alpha and/or associated withTNF-alpha (for example associated with an abnormal activity ofTNF-alpha, abnormal levels of TNF-alpha, abnormal expression ofTNF-alpha and/or abnormal sensitivity or response to TNF-alpha, or ofone of the biological phenomena associated with TNF-alpha), such as oneof the diseases or disorders mentioned above, which method comprisesadministering to a subject in need thereof a therapeutically activeamount of a Nanobody of the invention, of polypeptide of the invention,and/or of a pharmaceutical composition as described above.

The present invention provides polypeptides comprising one or morenanobodies directed towards tumor necrosis factor alpha (TNF-alpha). Thepresent invention further relates to their use in diagnosis and therapy.Such antibodies may have a framework sequence with high homology to thehuman framework sequences. Compositions comprising antibodies to tumornecrosis factor alpha (TNF-alpha) alone or in combination with otherdrugs are described.

Tumor necrosis factor alpha (TNF-alpha) is believed to play an importantrole in various disorders, for example in inflammatory disorders such asrheumatoid arthritis, Crohn's disease, ulcerative colitis and multiplesclerosis. Both TNF-alpha and the receptors (CD120a, CD120b) have beenstudied in great detail. TNF-alpha in its bioactive form is a trimer andthe groove formed by neighboring subunits is important for thecytokine-receptor interaction. Several strategies to antagonize theaction of the cytokine have been developed and are currently used totreat various disease states.

A TNF-alpha inhibitor which has sufficient specificity and selectivityto TNF-alpha may be an efficient prophylactic or therapeuticpharmaceutical compound for preventing or treating disorders whereTNF-alpha has been implicated as causative agent. Methods of treatingtoxic shock (EP 486526), tumor regression, inhibition of cytotoxicity(U.S. Pat. Nos. 6,448,380, 6,451,983, 6,498,237), autoimmune diseasesuch as RA and Crohn's disease (EP 663836, U.S. Pat. Nos. 5,672,347,5,656,272), graft versus host reaction (U.S. Pat. No. 5,672,347),bacterial meningitis (EP 585705) by means of an antibody to TNF-alphahave been described.

Yet none of the presently available drugs are completely effective forthe treatment of autoimmune disease, and most are limited by severetoxicity. In addition, it is extremely difficult and a lengthy processto develop a new chemical entitiy (NCE) with sufficient potency andselectivity to such target sequence. Antibody-based therapeutics on theother hand have significant potential as drugs because they haveexquisite specificity to their target and a low inherent toxicity. Inaddition, the development time can be reduced considerably when comparedto the development of new chemical entities (NCE's). However,conventional antibodies are difficult to raise against multimericproteins where the receptor-binding domain of the ligand is embedded ina groove, as is the case with TNF-alpha. Heavy chain antibodiesdescribed in the invention which are derived from Camelidae, are knownto have cavity-binding propensity (WO97/49805; Lauwereys et al, EMBO J.17, 5312, 1998)). Therefore, such heavy chain antibodies are inherentlysuited to bind to receptor binding domains of such ligands as TNF. Inaddition, such antibodies are known to be stable over long periods oftime, therefore increasing their shelf-life (Perez et al, Biochemistry,40, 74, 2001). Furthermore, such heavy chain antibody fragments can beproduced ‘en-masse’ in fermentors using cheap expression systemscompared to mammalian cell culture fermentation, such as yeast or othermicroorganisms (EP 0 698 097).

The use of antibodies derived from sources such as mouse, sheep, goat,rabbit etc., and humanised derivatives thereof as a treatment forconditions which require a modulation of inflammation is problematic forseveral reasons. Traditional antibodies are not stable at roomtemperature, and have to be refrigerated for preparation and storage,requiring necessary refrigerated laboratory equipment, storage andtransport, which contribute towards time and expense. Refrigeration issometimes not feasible in developing countries. Furthermore, themanufacture or small-scale production of said antibodies is expensivebecause the mammalian cellular systems necessary for the expression ofintact and active antibodies require high levels of support in terms oftime and equipment, and yields are very low. Furthermore the large sizeof conventional antibodies, would restrict tissue penetration, forexample, at the site of inflamed tissue. Furthermore, traditionalantibodies have a binding activity which depends upon pH, and hence areunsuitable for use in environments outside the usual physiological pHrange such as, for example, in treating gastric bleeding, gastricsurgery. Furthermore, traditional antibodies are unstable at low or highpH and hence are not suitable for oral administration. However, it hasbeen demonstrated that camelidae antibodies resist harsh conditions,such as extreme pH, denaturing reagents and high temperatures (Dumoulinet al, Protein Science 11, 500, 2002), so making them suitable fordelivery by oral administration. Furthermore, traditional antibodieshave a binding activity, which depends upon temperature, and hence areunsuitable for use in assays or kits performed at temperatures outsidebiologically active-temperature ranges (e.g. 37±20° C.).

Polypeptide therapeutics and in particular antibody-based therapeuticshave significant potential as drugs because they have exquisitespecificity to their target and a low inherent toxicity. However, it isknown by the skilled addressee that an antibody which has been obtainedfor a therapeutically useful target requires additional modification inorder to prepare it for human therapy, so as to avoid an unwantedimmunological reaction in a human individual upon administrationthereto. The modification process is commonly termed “humanisation”. Itis known by the skilled artisan that antibodies raised in species, otherthan in humans, require humanisation to render the antibodytherapeutically useful in humans ((1) CDR grafting: Protein Design Labs:U.S. Pat. Nos. 6,180,370, 5,693,761; Genentech U.S. Pat. No. 6,054,297;Celltech: 460167, EP 626390, U.S. Pat. No. 5,859,205; (2) Veneering:Xoma: U.S. Pat. Nos. 5,869,619, 5,766,886, 5,821,123). There is a needfor a method for producing antibodies which avoids the requirement forsubstantial humanisation, or which completely obviates the need forhumanisation. There is a need for a new class of antibodies which havedefined framework regions or amino acid residues and which can beadministered to a human subject without the requirement for substantialhumanisation, or the need for humanisation at all.

Another important drawback of conventional antibodies is that they arecomplex, large molecules and therefore relatively unstable, and they aresensitive to breakdown by proteases. This means that conventionalantibody drugs cannot be administered orally, sublingually, topically,nasally, vaginally, rectally or by inhalation because they are notresistant to the low pH at these sites, the action of proteases at thesesites and in the blood and/or because of their large size. They have tobe administered by injection (intravenously, subcutaneously, etc.) toovercome some of these problems. Administration by injection requiresspecialist training in order to use a hypodermic syringe or needlecorrectly and safely. It further requires sterile equipment, a liquidformulation of the therapeutic polypeptide, vial packing of saidpolypeptide in a sterile and stable form and, of the subject, a suitablesite for entry of the needle. Furthermore, subjects commonly experiencephysical and psychological stress prior to and upon receiving aninjection. Therefore, there is need for a method for the delivery oftherapeutic polypeptides which avoids the need for injection which isnot only cost/time saving, but which would also be more convenient andmore comfortable for the subject.

Nanobody-based therapeutics have significant potential as drugs becausethey have exquisite specificity to their target and a low inherenttoxicity. However, improving further their intrinsic and functionalaffinity can lead to many benefits for a patient such as reduced dose oftherapeutic, faster therapy, and reduced side effects.

One embodiment of the present invention is an anti-TNF-alpha nanobody,which nanobody is preferably as further defined above.

One embodiment of the present invention is an anti-TNF-alpha polypeptidecomprising at least one anti-TNF-alpha nanobody, which polypeptide ispreferably as further defined above.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above further comprising at least one nanobodydirected against a serum protein.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above wherein said serum protein is any ofserum albumin, serum immunoglobulins, thyroxine-binding protein,transferrin, or fibrinogen.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above further comprising at least one nanobodyselected from the group consisting of anti-IFN-gamma nanobody,anti-TNF-alpha receptor nanobody and anti-IFN-gamma receptor nanobody.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above, wherein the number of nanobodiesdirected against TNF-alpha is at least two.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above, wherein at least one nanobody is ahumanized Camelidae V_(HH)s.

Another embodiment of the present invention is a composition comprisingan anti-TNF-alpha polypeptide as described above and at least onenanobody from the group consisting of anti-IFN-gamma nanobody,anti-TNF-alpha receptor nanobody and anti-IFN-gamma receptor nanobody,for simultaneous, separate or sequential administration to a subject.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above, or a composition as described above,wherein said nanobody is an homologous sequence, a functional portion,or a functional portion of an homologous sequence of the full lengthnanobody.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above, or a composition as described above,wherein the anti-TNF-alpha polypeptide is an homologous sequence, afunctional portion, or a functional portion of an homologous sequence ofthe full length anti-TNF-alpha polypeptide.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above, or a composition as described abovewherein at least one nanobody is a Camelidae V_(HH).

Another embodiment of the present invention is a nucleic acid encodingan anti-TNF-alpha polypeptide as described above.

Another embodiment of the present invention is a method of identifyingan agent that modulates the binding of an anti-TNF-alpha polypeptide asdescribed above, to Tumor Necrosis Factor-alpha comprising the steps of:

(a) contacting an anti-TNF-alpha polypeptide as described above with atarget that is Tumor Necrosis Factor alpha, in the presence and absenceof a candidate modulator under conditions permitting binding betweensaid polypeptide and target, and

(b) measuring the binding between the polypeptide and target of step(a), wherein a decrease in binding in the presence of said candidatemodulator, relative to the binding in the absence of said candidatemodulator identified said candidate modulator as an agent that modulatesthe binding of an anti-TNF-alpha polypeptide as described above andTumor Necrosis Factor-alpha.

Another embodiment of the present invention is a method of identifyingan agent that modulates Tumor Necrosis Factor-alpha-mediated disordersthrough the binding of an anti-TNF-alpha polypeptide as described aboveto Tumor Necrosis Factor-alpha comprising:

(a) contacting an anti-TNF-alpha polypeptide as described above with atarget that is Tumor Necrosis Factor alpha, in the presence and absenceof a candidate modulator under conditions permitting binding betweensaid polypeptide and target, and

(b) measuring the binding between the polypeptide and target of step(a), wherein a decrease in binding in the presence of said candidatemodulator, relative to the binding in the absence of said candidatemodulator identified, said candidate modulator as an agent thatmodulates Tumor Necrosis Factor alpha-mediated disorders.

Another embodiment of the present invention is a method of identifyingan agent that modulates the binding of Tumor Necrosis Factor alpha toits receptor through the binding of an anti-TNF-alpha polypeptide asdescribed above to Tumor Necrosis Factor-alpha comprising:

(a) contacting an anti-TNF-alpha polypeptide as described above with atarget that is Tumor Necrosis Factor-alpha, in the presence and absenceof a candidate modulator under conditions permitting binding betweensaid polypeptide and target, and

(b) measuring the binding between the polypeptide and target of step(a), wherein a decrease in binding in the presence of said candidatemodulator, relative to the binding in the absence of said candidatemodulator identified said candidate modulator as an agent that modulatesthe binding of Tumor Necrosis Factor-alpha to its receptor.

Another embodiment of the present invention is a kit for screening foragents that modulate Tumor Necrosis Factor-alpha-mediated disorderscomprising an anti-TNF-alpha polypeptide as described above and TumorNecrosis Factor-alpha.

Another embodiment of the present invention is an unknown agent thatmodulates the binding of an anti-TNF-alpha polypeptide as describedabove to Tumor Necrosis Factor-alpha, identified according to the methodas described above.

Another embodiment of the present invention is an unknown agent thatmodulates Tumor Necrosis Factor-alpha-mediated disorders, identifiedaccording to the methods as described above.

Another embodiment of the present invention is an unknown agent asdescribed above wherein said disorders are one or more of inflammation,rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatorybowel syndrome and multiple sclerosis.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above, or a nucleic acid as described above, ora composition as described above, or an agent as described above fortreating and/or preventing and/or alleviating disorders relating toinflammatory processes.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as described above or a nucleic acid asdescribed above, or a composition as described above, or an agent asdescribed above for the preparation of a medicament for treating and/orpreventing and/or alleviating disorders relating to inflammatoryreactions.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above or a composition as described above, fortreating and/or preventing and/or alleviating disorders susceptible tomodulation by a Nanobody or polypeptide of the invention which is ablepass through the gastric environment without the substance beinginactivated.

Another embodiment of the present invention is an use of ananti-TNF-alpha polypeptide as described above or a composition asdescribed above, for the preparation of a medicament for treating,preventing and/or alleviating the symptoms of disorders susceptible tomodulation by a Nanobody or polypeptide of the invention which is ablepass through the gastric environment without the substance beinginactivated.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above or a composition as described above, fortreating and/or preventing and/or alleviating disorders susceptible tomodulation by a Nanobody or polypeptide of the invention delivered tothe vaginal and/or rectal tract.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as described above or a composition asdescribed above, for the preparation of a medicament for treating,preventing and/or alleviating the symptoms of disorders susceptible tomodulation by a Nanobody or polypeptide of the invention delivered tothe vaginal and/or rectal tract.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above or a composition as described above, fortreating and/or preventing and/or alleviating disorders susceptible tomodulation by a Nanobody or polypeptide of the invention delivered tothe nose, upper respiratory tract and/or lung.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as described above or a composition asdescribed above, for the preparation of a medicament for treating,preventing and/or alleviating the symptoms of disorders susceptible tomodulation by a Nanobody or polypeptide of the invention delivered tothe nose, upper respiratory tract and/or lung.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above or a composition as described above, fortreating and/or preventing and/or alleviating disorders susceptible tomodulation by a Nanobody or polypeptide of the invention delivered tothe intestinal mucosa, wherein said disorder increases the permeabilityof the intestinal mucosa.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as described above or a composition asdescribed above, for the preparation of a medicament for treating,preventing and/or alleviating the symptoms of disorders susceptible tomodulation by a Nanobody or polypeptide of the invention delivered tothe intestinal mucosa, wherein said disorder increases the permeabilityof the intestinal mucosa.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above or a composition as described above, fortreating and/or preventing and/or alleviating disorders susceptible tomodulation by a Nanobody or polypeptide of the invention which is ablepass through the tissues beneath the tongue effectively.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as described above or a composition asdescribed above, for the preparation of a medicament for treating,preventing and/or alleviating the symptoms of disorders susceptible tomodulation by a Nanobody or polypeptide of the invention which is ablepass through the tissues beneath the tongue effectively.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as described above or a composition as described above, fortreating and/or preventing and/or alleviating disorders susceptible tomodulation by a Nanobody or polypeptide of the invention which is ablepass through the skin effectively.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as described above or a composition asdescribed above, for the preparation of a medicament for treating,preventing and/or alleviating the symptoms of disorders susceptible tomodulation by a Nanobody or polypeptide of the invention which is ablepass through the skin effectively.

Another embodiment of the present invention is a method as describedabove, a kit as described above, a nucleic acid or agent as describedabove, use of a nucleic acid or agent as described above, a compositionas described above, use of a composition as described above, ananti-TNF-alpha polypeptide as described above, use of an anti-TNF-alphapolypeptide as described above wherein said disorders are any ofinflammation, rheumatoid arthritis, COPD, asthma, Crohn's disease,ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis,Addison's disease, Autoimmune hepatitis, Autoimmune parotitis, DiabetesType I, Epididymitis, Glomerulonephritis, Graves' disease,Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia, Systemiclupus erythematosus, Male infertility, Multiple sclerosis, MyastheniaGravis, Pemphigus, Psoriasis, Rheumatic fever, Rheumatoid arthritis,Sarcoidosis, Scleroderma, Sjogren's syndrome, Spondyloarthropathies,Thyroiditis, and Vasculitis.

Another embodiment of the present invention is a composition comprisinga nucleic acid or agent as described above, an anti-TNF-alphapolypeptide as described above, or a composition as described above, anda suitable pharmaceutical vehicle.

Another embodiment of the present invention is a method of diagnosing adisorder characterised by the dysfunction of Tumor Necrosis Factor-alphacomprising:

(a) contacting a sample with an anti-TNF-alpha polypeptide as describedabove,

(b) detecting binding of said polypeptide to said sample, and

(c) comparing the binding detected in step (b) with a standard, whereina difference in binding relative to said sample is diagnostic of adisorder characterised by dysfunction of Tumor Necrosis Factor-alpha.

Another embodiment of the present invention is a kit for screening for adisorder as cited above, using a method as described above.

Another embodiment of the present invention is a kit for screening for adisorder as cited above comprising an isolated anti-TNF-alphapolypeptide as described above.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as described above for the purification ofsaid Tumor Necrosis Factor-alpha.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as described above for inhibiting theinteraction between Tumor Necrosis Factor-alpha and one or more TumorNecrosis Factor-alpha receptors.

Another embodiment of the present invention is a method for producing ananti-TNF-alpha polypeptide as described above comprising the steps of:

(a) obtaining double stranded DNA encoding a Camelidae V_(HH) directedto Tumor Necrosis Factor alpha,

(b) cloning and expressing the DNA selected in step (b).

Another embodiment of the present invention is a method of producing ananti-TNF-alpha polypeptide as described above comprising:

(a) culturing host cells comprising nucleic acid capable of encoding ananti-TNF-alpha polypeptide as described above, under conditions allowingthe expression of the polypeptide, and,

(b) recovering the produced polypeptide from the culture.

Another embodiment of the present invention is a method as describedabove, wherein said host cells are bacterial or yeast.

Another embodiment of the present invention is a kit for screening forany of inflammation, rheumatoid arthritis, Crohn's disease, ulcerativecolitis, inflammatory bowel syndrome or multiple sclerosis comprising ananti-TNF-alpha polypeptide as described above.

V_(HH)s, according to the present invention, and as known to the skilledaddressee are heavy chain variable domains derived from immunoglobulinsnaturally devoid of light chains such as those derived from Camelidae asdescribed in WO 94/04678 (and referred to hereinafter as V_(HH) domainsor nanobodies). V_(HH) molecules are about 10× smaller than IgGmolecules. They are single polypeptides and very stable, resistingextreme pH and temperature conditions. Moreover, they are resistant tothe action of proteases which is not the case for conventionalantibodies. Furthermore, in vitro expression of V_(HH)s produces highyield, properly folded functional V_(HH)s. In addition, antibodiesgenerated in Camelids will recognize epitopes other than thoserecognised by antibodies generated in vitro through the use of antibodylibraries or via immunisation of mammals other than Camelids (WO9749805). As such, anti-TNF-alpha V_(HH)'s may interact more efficientlywith TNF-alpha than conventional antibodies, thereby blocking itsinteraction with the TNF-alpha receptor more efficiently.

TNF-alpha is also a fragment of TNF-alpha, capable of eliciting animmune response. TNF-alpha is also a fragment of TNF-alpha, capable ofbinding to a nanobody raised against the full length TNF-alpha.

A nanobody directed against TNF-alpha means nanobody that it is capableof binding to TNF-alpha with an affinity of better than 10⁻⁶ M.

One embodiment of the present invention is an anti-TNF polypeptide,wherein the nanobodies comprise Camelidae V_(HH) directed againstTNF-alpha.

The one or more nanobodies of the anti-TNF polypeptide which aredirected against a TNF-alpha may be of the same sequence. Alternativelythey may not all have the same sequence. It is within the scope of theinvention that an anti-TNF polypeptide comprises anti-TNF-alphananobodies which do not all share the same sequence, but which aredirected against the same target, one or more antigens thereof.

The present invention further relates to an anti-TNF-alpha polypeptide,wherein said nanobody is a V_(HH) directed against TNF-alpha, whereinthe V_(HH) belongs to a class having human-like sequences. The class ischaracterised in that the V_(HH)s carry an amino acid from the groupconsisting of glycine, alanine, valine, leucine, isoleucine, proline,phenylalanine, tyrosine, tryptophan, methionine, serine, threonine,asparagine, or glutamine at position 45, such as, for example, L45 and atryptophan at position 103, according to the Kabat numbering. Anotherhuman-like class of Camelidae nanobodies have been described inWO03035694 and contain the hydrophobic FR2 residues typically found inconventional antibodies of human origin or from other species, butcompensating this loss in hydrophilicity by the charged arginine residueon position 103 that substitutes the conserved tryptophan residuepresent in VH from double-chain antibodies. As such, peptides belongingto these two classes show a high amino acid sequence homology to humanVH framework regions and said peptides might be administered to a humandirectly without expectation of an unwanted immune response therefrom,and without the burden of further humanisation. The invention alsorelates to nucleic acids capable of encoding said polypeptides.

Any of the V_(HH)s as used by the invention may be of the traditionalclass or of the classes of human-like Camelidae antibodies. Saidantibodies may be directed against whole TNF-alpha or a fragmentthereof, or a fragment of a homologous sequence thereof. Thesepolypeptides include the full length Camelidae antibodies, namely Fc andV_(HH) domains, chimeric versions of heavy chain Camelidae antibodieswith a human Fc domain or V_(HH)'s by themselves or derived fragments.

Anti-serum albumin V_(HH)'s may interact in a more efficient way withserum albumin than conventional antibodies which is known to be acarrier protein. As a carrier protein some of the epitopes of serumalbumin may be inaccessible by bound proteins, peptides and smallchemical compounds. Since V_(HH)'s are known to bind into ‘unusual’ ornon-conventional epitopes such as cavities (WO 97/49805), the affinityof such V_(HH)'s to circulating albumin may be increased.

The present invention also relates to the finding that an anti-TNFpolypeptide as described herein further comprising one or morenanobodies directed against one or more serum proteins of a subject,surprisingly has significantly prolonged half-life in the circulation ofsaid subject compared with the half-life of the anti-TNF-alpha nanobodywhen not part of said construct. Furthermore, the said polypeptides werefound to exhibit the same favourable properties of nanobodies such ashigh stability remaining intact in mice, extreme pH resistance, hightemperature stability and high target affinity.

The serum protein may be any suitable protein found in the serum ofsubject. In one aspect of the invention, the serum protein is serumalbumin, serum immunoglobulins, thyroxine-binding protein, transferrin,or fibrinogen. Depending on the intended use such as the requiredhalf-life for effective treatment and/or compartimentalisation of thetarget antigen, the V_(HH)-partner can be directed to one of the aboveserum proteins.

According to a specific, but non-limiting aspect of the invention, theNanobody against human serum albumin consists of 4 framework regions(FR1 to FR4 respectively) and 3 complementarity determining regions(CDR1 to CDR3 respectively), in which:

-   (iv) CDR1 is an amino acid sequence chosen from the group consisting    of:

[SEQ ID NO: 36] SFGMS [SEQ ID NO: 37] LNLMG [SEQ ID NO: 38] INLLG[SEQ ID NO: 39] NYWMY;

-   -   and/or from the group consisting of amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences;        and in which:

-   (v) CDR2 is an amino acid sequence chosen from the group consisting    of:

[SEQ ID NO: 40] SISGSGSDTLYADSVKG [SEQ ID NO: 41] TITVGDSTNYADSVKG[SEQ ID NO: 42] TITVGDSTSYADSVKG [SEQ ID NO: 43] SINGRGDDTRYADSVKG[SEQ ID NO: 44] AISADSSTKNYADSVKG [SEQ ID NO: 45] AISADSSDKRYADSVKG[SEQ ID NO: 46] RISTGGGYSYYADSVKG

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences;        and in which:

-   (vi) CDR3 is an amino acid sequence chosen from the group consisting    of:

[SEQ ID NO: 47] DREAQVDTLDFDY

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences;    -   or from the group consisting of:

[SEQ ID NO: 48] GGSLSR [SEQ ID NO: 49] RRTWHSEL [SEQ ID NO: 50] GRSVSRS[SEQ ID NO: 51] GRGSP

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences.

In another aspect, the invention relates to a Nanobody against humanserum albumin, which consist of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), which is chosen from the group consisting of domainantibodies and/or single domain antibodies with the one of the followingcombinations of CDR1, CDR2 and CDR3, respectively:

CDR1: SFGMS; (SEQ ID NO: 36) CDR2: SISGSGSDTLYADSVKG; (SEQ ID NO: 40)CDR3: GGSLSR; (SEQ ID NO: 48) CDR1: LNLMG; (SEQ ID NO: 37)CDR2: TITVGDSTNYADSVKG; (SEQ ID NO: 41) CDR3: RRTWHSEL; (SEQ ID NO: 49)CDR1: INLLG; (SEQ ID NO: 38) CDR2: TITVGDSTSYADSVKG; (SEQ ID NO: 42)CDR3: RRTWHSEL; (SEQ ID NO: 49) CDR1: SFGMS; (SEQ ID NO: 36)CDR2: SINGRGDDTRYADSVKG; (SEQ ID NO: 43) CDR3: GRSVSRS; (SEQ ID NO: 50)CDR1: SFGMS; (SEQ ID NO: 36) CDR2: AISADSSDKRYADSVKG; (SEQ ID NO: 45)CDR3: GRGSP; (SEQ ID NO: 51) CDR1: SFGMS; (SEQ ID NO: 36)CDR2: AISADSSDKRYADSVKG; (SEQ ID NO: 45) CDR3: GRGSP; (SEQ ID NO: 51)CDR1: NYWMY; (SEQ ID NO: 39) CDR2: RISTGGGYSYYADSVKG; (SEQ ID NO: 46)CDR3: DREAQVDTLDFDY. (SEQ ID NO: 47)

In the Nanobodies of the invention that comprise the combinations ofCDR's mentioned above, each CDR can be replaced by a CDR chosen from thegroup consisting of amino acid sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with thementioned CDR's; in which

-   -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences;        and/or chosen from the group consisting of amino acid sequences        that have 3, 2 or only 1 (as indicated in the preceding        paragraph) “amino acid difference(s)” (as defined herein) with        the mentioned CDR(s) one of the above amino acid sequences, in        which:    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences.

However, of the Nanobodies of the invention that comprise thecombinations of CDR's mentioned above, Nanobodies comprising one or moreof the CDR's listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's listed above are more particularlypreferred; and Nanobodies comprising three of the CDR's listed above aremost particularly preferred.

In these Nanobodies against human serum albumin, the Framework regionsFR1 to FR4 are preferably as defined hereinabove for the Nanobodies ofthe invention.

Particularly preferred Nanobodies against human serum albumin are chosenfrom the group consisting of SEQ ID NO's: 61 to 67, SEQ ID NO's 87 to 89and SEQ ID NO's 100-104. The preferred combinations of CDR's andframework regions present in these Nanobodies are also listed in TableII

TABLE IIPreferred combination of Framework sequences and CDR's in Nanobodies against human serum albumin.Clone ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 PMP6A8 368AVQLVES 375 LNLMG 382 WYRQGPGN 389 TCITVGDSTNYA 396 RFTISMDYTKQTVYLHMN403 RRTWHSEL 410 WGQGTQV (ALB2) GGGLVQG ERELVA DSVKG SLRPEDTGLYYCKI TVSSGGSLRLAC AASERIFD PMP6B4 369 EVQLVES 376 INLLG 383 WYRQGPGN 390TITVGDSTSYAD 397 RFTISRDYDKNTLYLQMN 404 RRTWHSEL 411 WGQGTQV GGGLVQEGERELVA SVKG SLRPEDTGLYYCKI TVSS GSLRLACA ASERIWD PMP6A6 370 AVQLVES 377SFGMS 384 WVRQAPGK 391 SISGSGSDTLYA 398 RFTISRDNAKTTLYLQMN 405 GGSLSR412 SSQGTQV (ALB1) GGGLVQP EPEWVS DSVKG SLKPEDTAVYYCTI TVSS GNSLRLSCAASGFTFR PMP6C1 371 AVQLVDS 378 SFGMS 385 WVRQYPGK 392 SINGRGDDTRYA 399RFSISRDNAKNTLYLQMN 406 GRSVSRS 413 RTQGTQV GGGLVQPG EPEWVS DSVKGSLKPEDTAEYYCTI TVSS GSLRLSCA ASGFSFG PMP6G8 372 AVQLVESG 379 SFGMS 386WVRQAPGK 393 AISADSSTKNYA 400 RFTISRDNAKKMLYLEMN 407 GRGSP 414 SSPGTQVGGLVQPGG DQEWVS DSVKG SLKPEDTAVYYCVI TVSS SLRLTCTA SGFTFR PMP6A5 373QVQLAES 380 SFGMS 387 WVRQAPGE 394 AISADSSDKRYA 401 RFTISRDNAKKMLYLEMN408 GRGSP 415 ASQGTQV GGGLVQPG GLEWVS DSVKG SLKSEDTAVYYCVI TVSS GSLRLTCTASGFTFG PMP6G7 374 QVQLVESG 381 NYWMY 388 WVRVAPGK 395 RDISTGGGYSYY 402RFTISRDNAKNTLYLQMN 409 DREAQVDTLD 416 RGQGTQV GGLVQPGG GLERIS ADSVKGSLKPEDTALYYCAK FDY TVSS SLRLSCA ASGFTFSAnother aspect of the invention is an anti-TNF-alpha polypeptide asdisclosed herein further comprising at least one polypeptide selectedfrom the group consisting of an anti-IFN-gamma polypeptide, ananti-TNF-alpha receptor polypeptide and anti-IFN-gamma receptorpolypeptide.

According to one aspect of the invention, a nanobody is directed againstTNF-alpha receptor. Said nanobody may be a Camelidae V_(HH).

According to one aspect of the invention, a nanobody is directed againstIFN-gamma receptor. Said nanobody may be a Camelidae V_(HH).

Another aspect of the invention is a method of treating an autoimmunedisease or condition as cited herein, comprising administering to apatient an effective amount of an anti-TNF-alpha polypeptide furthercomprising a least one polypeptide selected from the group consisting ofanti-IFN-gamma polypeptide, anti-TNF-alpha receptor polypeptide andanti-IFN-gamma receptor polypeptide, such polypeptides joined to eachother as described below.

Such multi-specific constructs may have improved potency as inflammatorytherapeutic compound over mono-specific constructs.

One aspect of the invention is a composition comprising ananti-TNF-alpha polypeptide as disclosed herein and at least onepolypeptide selected from the group consisting of anti-IFN-gammapolypeptide, anti-TNF-alpha receptor polypeptide and anti-IFN-gammareceptor polypeptide, for simultaneous, separate or sequentialadministration to a subject.

One aspect of the invention is a method for treating autoimmune diseasecomprising administering to an individual an effective amount of ananti-TNF-alpha polypeptide and a least one polypeptide selected from thegroup consisting of anti-IFN-gamma polypeptide, anti-TNF-alpha receptorpolypeptide and anti-IFN-gamma receptor polypeptide, simultaneously,separately or sequentially.

Another aspect of the invention is a kit containing an anti-TNF-alphapolypeptide and a least one polypeptide selected from the groupconsisting of anti-IFN-gamma polypeptide, anti-TNF-alpha receptorpolypeptide and anti-IFN-gamma receptor polypeptide for simultaneous,separate or sequential administration to a subject. It is an aspect ofthe invention that the kit may be used according to the invention. It isan aspect of the invention that the kit may be used to treat thediseases as cited herein.

By simultaneous administration means the polypeptides are administeredto a subject at the same time. For example, as a mixture of thepolypeptides or a composition comprising said polypeptides. Examplesinclude, but are not limited to a solution administered intraveneously,a tablet, liquid, topical cream, etc., wherein each preparationcomprises the polypeptides of interest.

By separate administration means the polypeptides are administered to asubject at the same time or substantially the same time. Thepolypeptides are present in the kit as separate, unmixed preparations.For example, the different polypeptides may be present in the kit asindividual tablets. The tablets may be administered to the subject byswallowing both tablets at the same time, or one tablet directlyfollowing the other.

By sequential administration means the polypeptides are administered toa subject sequentially. The polypeptides are present in the kit asseparate, unmixed preparations. There is a time interval between doses.For example, one polypeptide might be administered up to 336, 312, 288,264, 240, 216, 192, 168, 144, 120, 96, 72, 48, 24, 20, 16, 12, 8, 4, 2,1, or 0.5 hours after the other component.

In sequential administration, one polypeptide may be administered once,or any number of times and in various doses before and/or afteradministration of another polypeptide. Sequential administration may becombined with simultaneous or sequential administration.

The medical uses of the anti-TNF-alpha polypeptide described below, alsoapply to the composition comprising an anti-TNF-alpha polypeptide asdisclosed herein and at least one polypeptide selected from the groupconsisting of anti-IFN-gamma polypeptide, anti-TNF-alpha receptorpolypeptide and anti-IFN-gamma receptor polypeptide, for simultaneous,separate or sequential administration to a subject as disclosed hereabove.

According to one aspect of the invention, an anti-IFN-gamma polypeptideanti-TNF-alpha a nanobody directed against IFN-gamma. Said nanobody maybe a Camelidae V_(HH).

According to one aspect of the invention, anti-TNF-alpha a nanobodydirected against TNF-alpha receptor. Said nanobody may be a CamelidaeV_(HH).

According to one aspect of the invention, an anti-IFN-gamma receptorpolypeptide anti-TNF-alpha a nanobody directed against IFN-gammareceptor. Said nanobody may be a Camelidae V_(HH).

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as disclosed herein, wherein the number of nanobodiesdirected against TNF-alpha is two or more. Such multivalentanti-TNF-alpha polypeptides have the advantage of unusually highfunctional affinity for the target, displaying much higher than expectedinhibitory properties compared to their monovalent counterparts.

The multivalent anti-TNF-alpha polypeptides have functional affinitiesthat are several orders of magnitude higher than the monovalent parentanti-TNF-alpha polypeptides. The inventors have found that thefunctional affinities of these multivalent polypeptides are much higherthan those reported in the prior art for bivalent and multivalentantibodies. Surprisingly, anti-TNF-alpha polypeptides of the presentinvention linked to each other directly or via a short linker sequenceshow the high functional affinities expected theoretically withmultivalent conventional four-chain antibodies.

The inventors have found that such large increased functional activitiescan be detected preferably with antigens composed of multidomain andmultimeric proteins, either in straight binding assays or in functionalassays, e.g. cytotoxicity assays.

The nanobodies may be joined to form any of the polypeptides disclosedherein comprising more than one nanobody using methods known in the artor any future method. For example, they may be fused by chemicalcross-linking by reacting amino acid residues with an organicderivatising agent such as described by Blattler et al, Biochemistry 24,1517-1524; EP294703. Alternatively, the nanobody may be fusedgenetically at the DNA level i.e. a polynucleotide construct formedwhich encodes the complete polypeptide construct comprising one or moreanti-target nanobodies and one or more anti-serum protein nanobodies. Amethod for producing bivalent or multivalent V_(HH) polypeptideconstructs is disclosed in PCT patent application WO 96/34103. One wayof joining multiple nanobodies is via the genetic route by linkingnanobody coding sequences either directly or via a peptide linker. Forexample, the C-terminal end of the first nanobody may be linked to theN-terminal end of the next nanobody. This linking mode can be extendedin order to link additional nanobodies for the construction andproduction of tri-, tetra-, etc. functional constructs.

According to one aspect of the present invention, the nanobodies arelinked to each other directly, without use of a linker. Contrary tojoining bulky conventional antibodies where a linker sequence is neededto retain binding activity in the two subunits, polypeptides of theinvention can be linked directly thereby avoiding potential problems ofthe linker sequence, such as antigenicity when administered to a humansubject, instability of the linker sequence leading to dissociation ofthe subunits.

According to another aspect of the present invention, the nanobodies arelinked to each other via a peptide linker sequence. Such linker sequencemay be a naturally occurring sequence or a non-naturally occurringsequence. The linker sequence is expected to be non-immunogenic in thesubject to which the anti-TNF-alpha polypeptide is administered. Thelinker sequence may provide sufficient flexibility to the multivalentanti-TNF-alpha polypeptide, at the same time being resistant toproteolytic degradation. A non-limiting example of a linker sequences isone that can be derived from the hinge region of V_(HH)s described in WO96/34103.

According to another aspect of the invention, multivalent nanobodiescomprising more than two nanobodies can be linked to each other eitherdirectly or via a linker sequence. Such constructs are difficult toproduce with conventional antibodies and due to steric hindrance of thebulky subunits, functionality will be lost or greatly diminished ratherthan increased considerably as seen with V_(HH)'s of the inventioncompared to the monovalent construct.

The polypeptide constructs disclosed herein may be made by the skilledartisan according to methods known in the art or any future method. Forexample, V_(HH)s may be obtained using methods known in the art such asby immunising a camel and obtaining hybridomas therefrom, or by cloninga library of nanobodies using molecular biology techniques known in theart and subsequent selection by using phage display.

According to an aspect of the invention an anti-TNF-alpha polypeptidemay be a homologous sequence of a full-length anti-TNF-alphapolypeptide. According to another aspect of the invention, ananti-TNF-alpha polypeptide may be a functional portion of a full-lengthanti-TNF-alpha polypeptide. According to another aspect of theinvention, an anti-TNF-alpha polypeptide may be a homologous sequence ofa full-length anti-TNF-alpha polypeptide. According to another aspect ofthe invention, an anti-TNF-alpha polypeptide may be a functional portionof a homologous sequence of a full-length anti-TNF-alpha polypeptide.According to an aspect of the invention an anti-TNF-alpha polypeptidemay comprise a sequence of an anti-TNF-alpha polypeptide.

According to an aspect of the invention a nanobody used to form ananti-TNF-alpha polypeptide may be a complete nanobody (e.g. a V_(HH)) ora homologous sequence thereof. According to another aspect of theinvention, a nanobody used to form the polypeptide construct may be afunctional portion of a complete nanobody. According to another aspectof the invention, a nanobody used to form the polypeptide construct maybe a homologous sequence of a complete nanobody. According to anotheraspect of the invention, a nanobody used to form the polypeptideconstruct may be a functional portion of a homologous sequence of acomplete nanobody.

As used herein, an homologous sequence of the present invention maycomprise additions, deletions or substitutions of one or more aminoacids, which do not substantially alter the functional characteristicsof the polypeptides of the invention. The number of amino acid deletionsor substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69 or 70 amino acids.

A homologous sequence according to the present invention may apolypeptide modified by the addition, deletion or substitution of aminoacids, said modification not substantially altering the functionalcharacteristics compared with the unmodified polypeptide.

A homologous sequence according to the present invention may be apolypeptide modified by the addition, deletion or substitution of aminoacids, said modification not substantially altering the functionalcharacteristics compared with the unmodified polypeptide.

A homologous sequence according to the present invention may be asequence which exists in other Camelidae species such as, for example,camel, dromedary, llama, alpaca, guanaco etc.

Where homologous sequence indicates sequence identity, it means asequence which presents a high sequence identity (more than 70%, 75%,80%, 85%, 90%, 95% or 98% sequence identity) with the parent sequenceand is preferably characterised by similar properties of the parentsequence, namely affinity, said identity calculated using known methods.

Alternatively, an homologous sequence may also be any amino acidsequence resulting from allowed substitutions at any number of positionsof the parent sequence according to the formula below:

Ser substituted by Ser, Thr, Gly, and Asn;

Arg substituted by one of Arg, His, Gln, Lys, and Glu;

Leu substituted by one of Leu, Ile, Phe, Tyr, Met, and Val;

Pro substituted by one of Pro, Gly, Ala, and Thr;

Thr substituted by one of Thr, Pro, Ser, Ala, Gly, His, and Gln;

Ala substituted by one of Ala, Gly, Thr, and Pro;

Val substituted by one of Val, Met, Tyr, Phe, Ile, and Leu;

Gly substituted by one of Gly, Ala, Thr, Pro, and Ser;

Ile substituted by one of Ile, Met, Tyr, Phe, Val, and Leu;

Phe substituted by one of Phe, Trp, Met, Tyr, Ile, Val, and Leu;

Tyr substituted by one of Tyr, Trp, Met, Phe, Ile, Val, and Leu;

His substituted by one of His, Glu, Lys, Gln, Thr, and Arg;

Gln substituted by one of Gln, Glu, Lys, Asn, His, Thr, and Arg;

Asn substituted by one of Asn, Glu, Asp, Gln, and Ser;

Lys substituted by one of Lys, Glu, Gln, His, and Arg;

Asp substituted by one of Asp, Glu, and Asn;

Glu substituted by one of Glu, Asp, Lys, Asn, Gln, His, and Arg;

Met substituted by one of Met, Phe, Ile, Val, Leu, and Tyr.

A homologous nucleotide sequence according to the present invention mayrefer to nucleotide sequences of more than 50, 100, 200, 300, 400, 500,600, 800 or 1000 nucleotides able to hybridize to the reverse-complementof the nucleotide sequence capable of encoding the patent sequence,under stringent hybridisation conditions (such as the ones described bySambrook et al., Molecular Cloning, Laboratory Manuel, Cold Spring,Harbor Laboratory press, New York).

As used herein, a functional portion refers to a sequence of a nanobodythat is of sufficient size such that the interaction of interest ismaintained with affinity of 1×10⁻⁶ M or better.

Alternatively, a functional portion comprises a partial deletion of thecomplete amino acid sequence and still maintains the binding site(s) andprotein domain(s) necessary for the binding of and interaction with thetarget.

As used herein, a functional portion refers to less than 100% of thecomplete sequence (e.g., 99%, 90%, 80%, 70%, 60% 50%, 40%, 30%, 20%,10%, 5%, 1% etc.), but comprises 5 or more amino acids or 15 or morenucleotides.

Targets as mentioned herein such as TNF-alpha, TNF-alpha receptor, serumproteins (e.g. serum albumin, serum immunoglobulins, thyroxine-bindingprotein, transferrin, fibrinogen) and IFN-gamma, IFN-gamma receptor maybe fragments of said targets. Thus a target is also a fragment of saidtarget, capable of eliciting an immune response. A target is also afragment of said target, capable of binding to a nanobody raised againstthe full length target.

A fragment as used herein refers to less than 100% of the sequence(e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), butcomprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 or more amino acids. A fragment is of sufficient lengthsuch that the interaction of interest is maintained with affinity of1×10⁻⁶ M or better.

A fragment as used herein also refers to optional insertions, deletionsand substitutions of one or more amino acids which do not substantiallyalter the ability of the target to bind to a nanobody raised against thewild-type target. The number of amino acid insertions deletions orsubstitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69 or 70 amino acids.

A homologous sequence of the present invention may include ananti-TNF-alpha polypeptide which has been humanised. The humanisation ofantibodies of the new class of V_(HH)s would further reduce thepossibility of unwanted immunological reaction in a human individualupon administration.

One embodiment of the present invention relates to a method forpreparing modified polypeptides based upon llama antibodies bydetermining the amino acid residues of the antibody variable domain(V_(HH)) which may be modified without diminishing the native affinityof the domain for antigen and while reducing its immunogenicity withrespect to a heterologous species; the use of V_(HH)s havingmodifications at the identified residues which are useful foradministration to heterologous species; and to the V_(HH) so modified.

More specifically, the invention relates to the preparation of modifiedV_(HH)s, which are modified for administration to humans, the resultingV_(HH) themselves, and the use of such “humanized” V_(HH)s in thetreatment of diseases in humans. By humanised is meant mutated so thatimmunogenicity upon administration in human patients is minor ornonexistent. Humanising a polypeptide, according to the presentinvention, comprises a step of replacing one or more of the Camelidaeamino acids by their human counterpart as found in the human consensussequence, without that polypeptide losing its typical character, i.e.the humanisation does not significantly affect the antigen bindingcapacity of the resulting polypeptide. Such methods are known by theskilled addressee.

Humanization of Camelidae nanobodies requires the introduction andmutagenesis of a limited amount of amino acids in a single polypeptidechain. This is in contrast to humanization of scFv, Fab, (Fab)2 and IgG,which requires the introduction of amino acid changes in two chains, thelight and the heavy chain and the preservation of the assembly of bothchains.

As described in WO 04/041862, an anti-TNF nanobody can be humanized.Humanization may for example involve mutagenesis of residues in FR1 atposition 1 and 5 which were introduced by the primer used for repertoirecloning and do not occur naturally in the llama sequence. Mutagenesis ofthose residues did not result in loss of binding and/or inhibitionactivity. Humanization may also involve mutagenesis of residues in FR3at position 74, 76, 83, 84, 93. Mutagenesis of those residues did notresult in a dramatic loss of binding and/or inhibition activity.Combining the mutations of FR1 and FR3 therefore did not affect thebinding and/or inhibition activity. Humanization may also involvemutagenesis of residues in FR4 at position 108. Mutagenesis of Q108Lresulted in lower production level in Escherichia coli. Position 108 issolvent exposed in camelid V_(HH), while in human antibodies thisposition is buried at the VH-VL interface (Spinelli, 1996; Nieba, 1997).In isolated VHs position 108 is solvent exposed. The introduction of anon-polar hydrophobic Leu instead of polar uncharged Gln can have adrastic effect on the intrinsic folding/stability of the molecule. Also,replacement of the hydrophilic residues by human hydrophobic residues atpositions 44 and 45 (E44G and R45L), did not have an effect on bindingand/or inhibition. However, loss of binding and/or inhibition activitywas observed when F37V and F47W were introduced. Modeling data confirmedthe critical residue 37 to preserve the integrity of the CDR3 loopconformation and hence on activity (all numbering according to theKabat).

According to one embodiment of the present invention, humanizationinvolves replacing of any of the following residues either alone or incombination:

FR1 position 1, 5, 28 and 30,

the hallmark amino acid at position 44 and 45 in FR2,

FR3 residues 74, 75, 76, 83, 84, 93 and 94,

and positions 103, 104, 108 and 111 in FR4;

numbering according to the Kabat numbering.

One embodiment of the present invention is an anti-TNF-alphapolypeptide, or a nucleic acid capable of encoding said polypeptide foruse in treating, preventing and/or alleviating the symptoms of disordersrelating to inflammatory processes. TNF-alpha is involved ininflammatory processes, and the blocking of TNF-alpha action can have ananti-inflammatory effect, which is highly desirable in certain diseasestates such as, for example, Crohn's disease. The Examples demonstrateV_(HH)s according to the invention which bind TNF-alpha and moreover,block its binding to the TNF-alpha receptor.

The anti-TNF-alpha polypeptides of the present invention are applicableto autoimmune diseases, such as Addison's disease (adrenal), Autoimmunediseases of the ear (ear), Autoimmune diseases of the eye (eye),Autoimmune hepatitis (liver), Autoimmune parotitis (parotid glands),Crohn's disease (intestine), Diabetes Type I (pancreas), Epididymitis(epididymis), Glomerulonephritis (kidneys), Graves' disease (thyroid),Guillain-Barre syndrome (nerve cells), Hashimoto's disease (thyroid),Hemolytic anemia (red blood cells), Systemic lupus erythematosus(multiple tissues), Male infertility (sperm), Multiple sclerosis (nervecells), Myasthenia Gravis (neuromuscular junction), Pemphigus (primarilyskin), Psoriasis (skin), Rheumatic fever (heart and joints), Rheumatoidarthritis (joint lining), Sarcoidosis (multiple tissues and organs),Scleroderma (skin and connective tissues), Sjogren's syndrome (exocrineglands, and other tissues), Spondyloarthropathies (axial skeleton, andother tissues), Thyroiditis (thyroid), Vasculitis (blood vessels).

Within parenthesis is the tissue affected by the disease. This listingof autoimmune diseases is intended to be exemplary rather thaninclusive.

Autoimmune conditions for which the anti-TNF-alpha polypeptides of thepresent invention is applicable include, for example, AIDS, atopicallergy, bronchial asthma, eczema, leprosy, schizophrenia, inheriteddepression, transplantation of tissues and organs, chronic fatiguesyndrome, Alzheimer's disease, Parkinson's disease, myocardialinfarction, stroke, autism, epilepsy, Arthus's phenomenon, anaphylaxis,and alcohol and drug addiction. In the above-identified autoimmuneconditions, the tissue affected is the primary target, in other cases itis the secondary target. These conditions are partly or mostlyautoimmune syndromes. Therefore, in treating them, it is possible to usethe same methods, or aspects of the same methods that are hereindisclosed, sometimes in combination with other methods.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide according to the invention, or a nucleic acidcapable of encoding said polypeptide for the preparation of a medicamentfor treating a disorder relating to inflammatory processes. Examples ofdisorders include rheumatoid arthritis, Crohn's disease, ulcerativecolitis, inflammatory bowel syndrome and multiple sclerosis.

Polypeptides and nucleic acids according to the present invention may beadministered to a subject by conventional routes, such as intravenously.However, a special property of the anti-TNF-alpha polypeptides of theinvention is that they penetrate barriers such as tissue membranesand/or tumours and act locally thereon, and they are sufficiently stableto withstand extreme environments such as in the stomach. Therefore,another aspect of the present invention relates to the delivery ofanti-TNF-alpha polypeptides.

When the Nanobodies and/or polypeptides of the invention are used for,or are intended for use in, the prevention or treatment of diseases anddisorders of the gastro-intestinal tract, in particular by means of oraladministration or other administration into the gastrointestinal tract,it will usually not be necessary to use polypeptides of the inventionthat have increased half-life in serum (i.e. that have been pegylated orthat contain a Nanobody directed against a serum protein). Thus, forsuch indications, polypeptides of the invention can be used that onlycontain Nanobodies of the invention. In particular, it has been foundthat for oral administration for the prevention and treatment ofdiseases or disorders of the gastro-intestinal tract associated withand/or mediated by TNF-alpha (such as IBD and the other diseases anddisorders of the gastro-intestinal tract mentioned above), the use of amonovalent Nanobody of the invention or of a polypeptide of theinvention that essentially consists of a monovalent Nanobody of theinvention may be preferred. For other indications, such as the treatmentof rheumatoid arthritis (RA), the use of a bivalent Nanobody of theinvention may be preferred. When such a Nanobody has to reach itsintended site of action via the blood stream, the use of a polypeptideof the invention that has increased half-life in serum may be preferred.

A subject according to the invention can be any mammal susceptible totreatment by therapeutic polypeptides.

Oral delivery of anti-TNF-alpha polypeptides of the invention results inthe provision of such molecules in an active form in the colon at localsites that are affected by the disorder. These sites may be highlyinflamed and contain TNF-alpha-producing cells. The anti-TNF-alphapolypeptides of the invention which bind to TNF-alpha can neutralise theTNF-alpha locally, avoiding distribution throughout the whole body andthus limiting negative side-effects. Genetically modified microorganismssuch as Micrococcus lactis are able to secrete antibody or functionalportions thereof. Such modified microorganisms can be used as vehiclesfor local production and delivery of antibodies or functional portionsthereof in the intestine. By using a strain which produces ananti-TNF-alpha polypeptide, inflammatory bowel syndrome could betreated.

Another aspect of the invention involves delivering anti-TNFpolypeptides by using surface expression on or secretion fromnon-invasive bacteria, such as Gram-positive host organisms likeLactococcus spec. using a vector such as described in WO00/23471.

One embodiment of the present invention is an anti-TNF-alpha polypeptideas disclosed herein for use in treating, preventing and/or alleviatingthe symptoms of disorders susceptible to modulation by a Nanobody orpolypeptide of the invention which is able pass through the gastricenvironment without the substance being inactivated.

Examples of disorders are any that cause inflammation, including, butnot limited to rheumatoid arthritis, Crohn's disease, ulcerativecolitis, inflammatory bowel syndrome, and multiple sclerosis. As knownby persons skilled in the art, once in possession of said polypeptideconstruct, formulation technology may be applied to release a maximumamount of polypeptide in the right location (in the stomach, in thecolon, etc.). This method of delivery is important for treating, preventand/or alleviate the symptoms of disorders whose targets are located inthe gut system.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of a disorder susceptible to modulation by aNanobody or polypeptide of the invention which is able pass through thegastric environment without being inactivated, by orally administeringto a subject an anti-TNF-alpha polypeptide as disclosed herein.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as disclosed herein for the preparation of amedicament for treating, preventing and/or alleviating the symptoms ofdisorders susceptible to modulation by a Nanobody or polypeptide of theinvention which is able pass through the gastric environment withoutbeing inactivated.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the gut system without said substancebeing inactivated, by orally administering to a subject ananti-TNF-alpha polypeptide as disclosed herein.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the bloodstream of a subject without thesubstance being inactivated, by orally administering to a subject ananti-TNF-alpha polypeptide as disclosed herein.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as disclosed herein for use in treating, preventing and/oralleviating the symptoms or disorders susceptible to modulation by aNanobody or polypeptide of the invention delivered to the vaginal and/orrectal tract.

Examples of disorders are any that cause inflammation, including, butnot limited to rheumatoid arthritis, Crohn's disease, ulcerativecolitis, inflammatory bowel syndrome, and multiple sclerosis. In anon-limiting example, a formulation according to the invention comprisesan anti-TNF-alpha polypeptide as disclosed herein, in the form of a gel,cream, suppository, film, or in the form of a sponge or as a vaginalring that slowly releases the active ingredient over time (suchformulations are described in EP 707473, EP 684814, U.S. Pat. No.5,629,001).

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by aNanobody or polypeptide of the invention delivered to the vaginal and/orrectal tract, by vaginally and/or rectally administering to a subject ananti-TNF-alpha polypeptide as disclosed herein.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as disclosed herein for the preparation of amedicament for treating, preventing and/or alleviating the symptoms ofdisorders susceptible to modulation by a Nanobody or polypeptide of theinvention delivered to the vaginal and/or rectal tract.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the vaginal and/or rectal tract withoutbeing said substance being inactivated, by administering to the vaginaland/or rectal tract of a subject an anti-TNF-alpha polypeptide asdisclosed herein.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the bloodstream of a subject withoutsaid substance being inactivated, by administering to the vaginal and/orrectal tract of a subject an anti-TNF-alpha polypeptide as disclosedherein.

Another embodiment of the present invention is an anti-TNF-alphapolypeptide as disclosed herein, for use in treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by aNanobody or polypeptide of the invention delivered to the nose, upperrespiratory tract and/or lung.

Examples of disorders are any that cause inflammation, including, butnot limited to rheumatoid arthritis, Crohn's disease, ulcerativecolitis, inflammatory bowel syndrome, and multiple sclerosis. In anon-limiting example, a formulation according to the invention,comprises an anti-TNF-alpha polypeptide as disclosed herein in the formof a nasal spray (e.g. an aerosol) or inhaler. Since the polypeptideconstruct is small, it can reach its target much more effectively thantherapeutic IgG molecules.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by aNanobody or polypeptide of the invention delivered to the upperrespiratory tract and lung, by administering to a subject ananti-TNF-alpha polypeptide as disclosed herein, by inhalation throughthe mouth or nose.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as disclosed herein for the preparation of amedicament for treating, preventing and/or alleviating the symptoms ofdisorders susceptible to modulation by a Nanobody or polypeptide of theinvention delivered to the nose, upper respiratory tract and/or lung,without said polypeptide being inactivated.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the nose, upper respiratory tract andlung without inactivation, by administering to the nose, upperrespiratory tract and/or lung of a subject an anti-TNF-alpha polypeptideas disclosed herein.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the bloodstream of a subject withoutinactivation by administering to the nose, upper respiratory tractand/or lung of a subject an anti-TNF-alpha polypeptide as disclosedherein.

One embodiment of the present invention is an anti-TNF-alpha polypeptideas disclosed herein for use in treating, preventing and/or alleviatingthe symptoms of disorders susceptible to modulation by a Nanobody orpolypeptide of the invention delivered to the intestinal mucosa, whereinsaid disorder increases the permeability of the intestinal mucosa.Because of their small size, an anti-TNF-alpha polypeptide as disclosedherein can pass through the intestinal mucosa and reach the bloodstreammore efficiently in subjects suffering from disorders which cause anincrease in the permeability of the intestinal mucosa, for exampleCrohn's disease.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by aNanobody or polypeptide of the invention delivered to the intestinalmucosa, wherein said disorder increases the permeability of theintestinal mucosa, by orally administering to a subject ananti-TNF-alpha polypeptide as disclosed herein.

This process can be even further enhanced by an additional aspect of thepresent invention—the use of active transport carriers. In this aspectof the invention, V_(HH) is fused to a carrier that enhances thetransfer through the intestinal wall into the bloodstream. In anon-limiting example, this “carrier” is a second V_(HH) which is fusedto the therapeutic V_(HH). Such fusion constructs are made using methodsknown in the art. The “carrier” V_(HH) binds specifically to a receptoron the intestinal wall which induces an active transfer through thewall.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as disclosed herein for the preparation of amedicament for treating, preventing and/or alleviating the symptoms ofdisorders susceptible to modulation by a Nanobody or polypeptide of theinvention delivered to the intestinal mucosa, wherein said disorderincreases the permeability of the intestinal mucosa.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the intestinal mucosa without beinginactivated, by administering orally to a subject an anti-TNF-alphapolypeptide of the invention.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the bloodstream of a subject withoutbeing inactivated, by administering orally to a subject ananti-TNF-alpha polypeptide of the invention.

This process can be even further enhanced by an additional aspect of thepresent invention—the use of active transport carriers. In this aspectof the invention, an anti-TNF-alpha polypeptide as described herein isfused to a carrier that enhances the transfer through the intestinalwall into the bloodstream. In a non-limiting example, this “carrier” isa V_(HH) which is fused to said polypeptide. Such fusion constructs madeusing methods known in the art. The “carrier” V_(HH) binds specificallyto a receptor on the intestinal wall which induces an active transferthrough the wall.

One embodiment of the present invention is an anti-TNF-alpha polypeptideas disclosed herein for use in treating, preventing and/or alleviatingthe symptoms of disorders susceptible to modulation by a Nanobody orpolypeptide of the invention which is able pass through the tissuesbeneath the tongue effectively.

Examples of disorders are any that cause inflammation, including, butnot limited to rheumatoid arthritis, Crohn's disease, ulcerativecolitis, inflammatory bowel syndrome, and multiple sclerosis. Aformulation of said polypeptide construct as disclosed herein, forexample, a tablet, spray, drop is placed under the tongue and adsorbedthrough the mucus membranes into the capillary network under the tongue.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by aNanobody or polypeptide of the invention which is able pass through thetissues beneath the tongue effectively, by sublingually administering toa subject an anti-TNF-alpha polypeptide as disclosed herein.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as disclosed herein for the preparation of amedicament for treating, preventing and/or alleviating the symptoms ofdisorders susceptible to modulation by a Nanobody or polypeptide of theinvention which is able to pass through the tissues beneath the tongue.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the tissues beneath the tongue withoutbeing inactivated, by administering sublingually to a subject ananti-TNF-alpha polypeptide as disclosed herein.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the bloodstream of a subject withoutbeing inactivated, by administering orally to a subject ananti-TNF-alpha polypeptide as disclosed herein.

One embodiment of the present invention is an anti-TNF-alpha polypeptideas disclosed herein for use in treating, preventing and/or alleviatingthe symptoms of disorders susceptible to modulation by a Nanobody orpolypeptide of the invention which is able pass through the skineffectively.

Examples of disorders are any that cause inflammation, including, butnot limited to rheumatoid arthritis, Crohn's disease, ulcerativecolitis, inflammatory bowel syndrome, and multiple sclerosis. Aformulation of said polypeptide construct, for example, a cream, film,spray, drop, patch, is placed on the skin and passes through.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by aNanobody or polypeptide of the invention which is able pass through theskin effectively, by topically administering to a subject ananti-TNF-alpha polypeptide as disclosed herein.

Another embodiment of the present invention is a use of ananti-TNF-alpha polypeptide as disclosed herein for the preparation of amedicament for treating, preventing and/or alleviating the symptoms ofdisorders susceptible to modulation by a Nanobody or polypeptide of theinvention which is able pass through the skin effectively.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the skin without being inactivated, byadministering topically to a subject an anti-TNF-alpha polypeptide asdisclosed herein.

An aspect of the invention is a method for delivering a Nanobody orpolypeptide of the invention to the bloodstream of a subject, byadministering topically to a subject an anti-TNF-alpha polypeptide asdisclosed herein.

In another embodiment of the present invention, an anti-TNF-alphapolypeptide further comprises a carrier nanobody (e.g. V_(HH)) whichacts as an active transport carrier for transport said anti-TNF-alphapolypeptide, from the lung lumen to the blood.

An anti-TNF-alpha polypeptide further comprising a carrier bindsspecifically to a receptor present on the mucosal surface (bronchialepithelial cells) resulting in the active transport of the polypeptidefrom the lung lumen to the blood. The carrier nanobody may be fused tothe polypeptide construct. Such fusion constructs may be made usingmethods known in the art and are describe herein. The “carrier” nanobodybinds specifically to a receptor on the mucosal surface which induces anactive transfer through the surface.

Another aspect of the present invention is a method to determine whichnanobodies (e.g. V_(HH)s) are actively transported into the bloodstreamupon nasal administration. Similarly, a naïve or immune V_(HH) phagelibrary can be administered nasally, and after different time pointsafter administration, blood or organs can be isolated to rescue phagesthat have been actively transported to the bloodstream. A non-limitingexample of a receptor for active transport from the lung lumen to thebloodstream is the Fc receptor N (FcRn). One aspect of the inventionincludes the V_(HH) molecules identified by the method. Such V_(HH) canthen be used as a carrier V_(HH) for the delivery of a therapeuticV_(HH) to the corresponding target in the bloodstream upon nasaladministration.

In one aspect of the invention, one can use an anti-TNF-alphapolypeptide as disclosed herein, in order to screen for agents thatmodulate the binding of the polypeptide to TNF-alpha. When identified inan assay that measures binding or said polypeptide displacement alone,agents will have to be subjected to functional testing to determinewhether they would modulate the action of the antigen in vivo.

In an example of a displacement experiment, phage or cells expressingTNF-alpha or a fragment thereof are incubated in binding buffer withpolypeptide of the invention which has been labeled, in the presence orabsence of increasing concentrations of a candidate modulator. Tovalidate and calibrate the assay, control competition reactions usingincreasing concentrations of said polypeptide and which is unlabeled,can be performed. After incubation, cells are washed extensively, andbound, labeled polypeptide is measured as appropriate for the givenlabel (e.g., scintillation counting, fluorescence, etc.). A decrease ofat least 10% in the amount of labeled polypeptide bound in the presenceof candidate modulator indicates displacement of binding by thecandidate modulator. Candidate modulators are considered to bindspecifically in this or other assays described herein if they displace50% of labeled polypeptide (sub-saturating polypeptide dose) at aconcentration of 1 μM or less.

Alternatively, binding or displacement of binding can be monitored bysurface plasmon resonance (SPR). Surface plasmon resonance assays can beused as a quantitative method to measure binding between two moleculesby the change in mass near an immobilized sensor caused by the bindingor loss of binding of the polypeptide of the invention from the aqueousphase to TNF-alpha immobilized in a membrane on the sensor. This changein mass is measured as resonance units versus time after injection orremoval of the said polypeptide or candidate modulator and is measuredusing a Biacore Biosensor (Biacore AB). TNF-alpha can be for exampleimmobilized on a sensor chip (for example, research grade CM5 chip;Biacore AB) in a thin film lipid membrane according to methods describedby Salamon et al. (Salamon et al., 1996, Biophys J. 71: 283-294; Salamonet al., 2001, Biophys. J. 80: 1557-1567; Salamon et al., 1999, TrendsBiochem. Sci. 24: 213-219, each of which is incorporated herein byreference.). Sarrio et al. demonstrated that SPR can be used to detectligand binding to the GPCR A(1) adenosine receptor immobilized in alipid layer on the chip (Sarrio et al., 2000, Mol. Cell. Biol. 20:5164-5174, incorporated herein by reference). Conditions for the bindingof a polypeptide of the invention to TNF-alpha in an SPR assay can befine-tuned by one of skill in the art using the conditions reported bySarrio et al. as a starting point.

SPR can assay for modulators of binding in at least two ways. First, apolypeptide of the invention can be pre-bound to immobilized TNF-alphafollowed by injection of candidate modulator at a concentration rangingfrom 0.1 nM to 1 μM. Displacement of the bound polypeptide can bequantitated, permitting detection of modulator binding. Alternatively,the membrane-bound TNF-alpha can be pre-incubated with a candidatemodulator and challenged with the polypeptide of the invention. Adifference in binding affinity between said polypeptide and TNF-alphapre-incubated with the modulator, compared with that between saidpolypeptide and TNF-alpha in absence of the modulator will demonstratebinding or displacement of said polypeptide in the presence ofmodulator. In either assay, a decrease of 10% or more in the amount ofsaid polypeptide bound in the presence of candidate modulator, relativeto the amount of said polypeptide bound in the absence of candidatemodulator indicates that the candidate modulator inhibits theinteraction of TNF-alpha and said polypeptide.

Another method of detecting inhibition of binding of, for example, apolypeptide of the invention, to TNF-alpha uses fluorescence resonanceenergy transfer (FRET). FRET is a quantum mechanical phenomenon thatoccurs between a fluorescence donor (D) and a fluorescence acceptor (A)in close proximity to each other (usually <100 Å of separation) if theemission spectrum of D overlaps with the excitation spectrum of A. Themolecules to be tested, e.g. a polypeptide polypeptide of the inventionand a TNF-alpha are labelled with a complementary pair of donor andacceptor fluorophores. While bound closely together by the TNF-alpha:polypeptide interaction, the fluorescence emitted upon excitation of thedonor fluorophore will have a different wavelength from that emitted inresponse to that excitation wavelength when the said polypeptide andTNF-alpha are not bound, providing for quantitation of bound versusunbound molecules by measurement of emission intensity at eachwavelength. Donor fluorophores with which to label the TNF-alpha arewell known in the art. Of particular interest are variants of the A.Victoria GFP known as Cyan FP (CFP, Donor (D)) and Yellow FP (YFP,Acceptor (A)). As an example, the YFP variant can be made as a fusionprotein with TNF-alpha. Vectors for the expression of GFP variants asfusions (Clontech) as well as fluorophore-labeled reagents (MolecularProbes) are known in the art. The addition of a candidate modulator tothe mixture of fluorescently-labelled polypeptide and YFP-TNF-alpha willresult in an inhibition of energy transfer evidenced by, for example, adecrease in YFP fluorescence relative to a sample without the candidatemodulator. In an assay using FRET for the detection of TNF-alpha:polypeptide interaction, a 10% or greater decrease in the intensity offluorescent emission at the acceptor wavelength in samples containing acandidate modulator, relative to samples without the candidatemodulator, indicates that the candidate modulator inhibits theTNF-alpha:polypeptide interaction.

A sample as used herein may be any biological sample containingTNF-alpha such as clinical (e.g. cell fractions, whole blood, plasma,serum, tissue, cells, etc.), derived from clinical, agricultural,forensic, research, or other possible samples. The clinical samples maybe from human or animal origin. The sample analysed can be both solid orliquid in nature. It is evident when solid materials are used, these arefirst dissolved in a suitable solution.

A variation on FRET uses fluorescence quenching to monitor molecularinteractions. One molecule in the interacting pair can be labelled witha fluorophore, and the other with a molecule that quenches thefluorescence of the fluorophore when brought into close apposition withit. A change in fluorescence upon excitation is indicative of a changein the association of the molecules tagged with the fluorophore:quencherpair. Generally, an increase in fluorescence of the labelled TNF-alphais indicative that anti-TNF-alpha polypeptide bearing the quencher hasbeen displaced. For quenching assays, a 10% or greater increase in theintensity of fluorescent emission in samples containing a candidatemodulator, relative to samples without the candidate modulator,indicates that the candidate modulator inhibits TNF-alpha:anti-TNF-alpha polypeptide interaction.

In addition to the surface plasmon resonance and FRET methods,fluorescence polarization measurement is useful to quantitate binding.The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Complexes,such as those formed by TNF-alpha associating with a fluorescentlylabelled anti-TNF-alpha polypeptide, have higher polarization valuesthan uncomplexed, labelled polypeptide. The inclusion of a candidateinhibitor of the TNF-alpha:anti-TNF-alpha polypeptide interactionresults in a decrease in fluorescence polarization, relative to amixture without the candidate inhibitor, if the candidate inhibitordisrupts or inhibits the interaction of TNF-alpha with said polypeptide.Fluorescence polarization is well suited for the identification of smallmolecules that disrupt the formation of TNF-alpha:anti-TNF-alphapolypeptide complexes. A decrease of 10% or more in fluorescencepolarization in samples containing a candidate modulator, relative tofluorescence polarization in a sample lacking the candidate modulator,indicates that the candidate modulator inhibits the TNF-alpha:anti-TNF-alpha polypeptide interaction.

Another alternative for monitoring TNF-alpha: anti-TNF-alpha polypeptideinteractions uses a biosensor assay. ICS biosensors have been describedin the art (Australian Membrane Biotechnology Research Institute;Cornell B, Braach-Maksvytis V, King L, Osman P, Raguse B, Wieczorek L,and Pace R. “A biosensor that uses ion-channel switches” Nature 1997,387, 580). In this technology, the association of TNF-alpha and aanti-TNF-alpha polypeptide is coupled to the closing ofgramicidine-facilitated ion channels in suspended membrane bilayers andthus to a measurable change in the admittance (similar to impedance) ofthe biosensor. This approach is linear over six orders of magnitude ofadmittance change and is ideally suited for large scale, high throughputscreening of small molecule combinatorial libraries. A 10% or greaterchange (increase or decrease) in admittance in a sample containing acandidate modulator, relative to the admittance of a sample lacking thecandidate modulator, indicates that the candidate modulator inhibits theinteraction of TNF-alpha and said polypeptide. It is important to notethat in assays testing the interaction of TNF-alpha with ananti-TNF-alpha polypeptide, it is possible that a modulator of theinteraction need not necessarily interact directly with the domain(s) ofthe proteins that physically interact with said polypeptide. It is alsopossible that a modulator will interact at a location removed from thesite of interaction and cause, for example, a conformational change inthe TNF-alpha. Modulators (inhibitors or agonists) that act in thismanner are nonetheless of interest as agents to modulate the binding ofTNF-alpha to its receptor.

Any of the binding assays described can be used to determine thepresence of an agent in a sample, e.g., a tissue sample, that binds toTNF-alpha, or that affects the binding of, for example, a polypeptidepolypeptide of the invention to the TNF-alpha. To do so a TNF-alpha isreacted with said polypeptide in the presence or absence of the sample,and polypeptide binding is measured as appropriate for the binding assaybeing used. A decrease of 10% or more in the binding of said polypeptideindicates that the sample contains an agent that modulates the bindingof said polypeptide to the TNF-alpha. Of course, the above-generalizedmethod might easily be applied to screening for candidate modulatorswhich alter the binding between any anti-TNF-alpha polypeptide of theinvention, an homologous sequence thereof, a functional portion thereofor a functional portion of an homologous sequence thereof, and TNF-alphaor a fragment thereof.

One embodiment of the present invention is an unknown agent identifiedby the method disclosed herein.

One embodiment of the present invention is an unknown agent identifiedby the method disclosed herein for use in treating, preventing and/oralleviating the symptoms of disorders relating to inflammatoryprocesses.

Another embodiment of the present invention is a use of an unknown agentidentified by the method disclosed herein for use in treating,preventing and/or alleviating the symptoms of disorders relating toinflammatory processes.

Examples of disorders include rheumatoid arthritis, Crohn's disease,ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.

A cell that is useful according to the invention is preferably selectedfrom the group consisting of bacterial cells such as, for example, E.coli, yeast cells such as, for example, S. cerevisiae, P. pastoris,insect cells or mammal cells.

A cell that is useful according to the invention can be any cell intowhich a nucleic acid sequence encoding a polypeptide comprising ananti-TNF-alpha of the invention, an homologous sequence thereof, afunctional portion thereof or a functional portion of an homologoussequence thereof according to the invention can be introduced such thatthe polypeptide is expressed at natural levels or above natural levels,as defined herein. Preferably a polypeptide of the invention that isexpressed in a cell exhibits normal or near normal pharmacology, asdefined herein.

According to a preferred embodiment of the present invention, a cell isselected from the group consisting of COS7-cells, a CHO cell, a LM (TK-)cell, a NIH-3T3 cell, HEK-293 cell, K-562 cell or a 1321N1 astrocytomacell but also other transfectable cell lines.

In general, “therapeutically effective amount”, “therapeuticallyeffective dose” and “effective amount” means the amount needed toachieve the desired result or results (modulating TNF-alpha binding;treating or preventing inflammation). One of ordinary skill in the artwill recognize that the potency and, therefore, an “effective amount”can vary for the various compounds that modulate TNF-alpha binding usedin the invention. One skilled in the art can readily assess the potencyof the compound.

As used herein, the term “compound” refers to an anti-TNF-alphapolypeptide of the present invention, a composition, or a nucleic acidcapable of encoding said polypeptide or an agent identified according tothe screening method described herein or said polypeptide comprising oneor more derivatised amino acids.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the compound without causingany undesirable biological effects or interacting in a deleteriousmanner with any of the other components of the pharmaceuticalcomposition in which it is contained.

Anti-TNF-alpha polypeptides as disclosed herein is useful for treatingor preventing conditions in a subject and comprises administering apharmaceutically effective amount of a compound or composition.

Anti-TNF polypeptides of the present invention are useful for treatingor preventing conditions relating to rheumatoid arthritis, Crohn'sdisease, ulcerative colitis, inflammatory bowel syndrome and multiplesclerosis in a subject and comprises administering a pharmaceuticallyeffective amount of a compound or composition that binds TNF-alpha.

Anti-TNF-alpha polypeptides as disclosed here in are useful for treatingor preventing conditions in a subject and comprises administering apharmaceutically effective amount of a compound combination withanother, such as, for example, aspirin.

The anti-TNF-alpha polypeptides as disclosed here in are useful fortreating or preventing conditions relating to rheumatoid arthritis,Crohn's disease, ulcerative colitis, inflammatory bowel syndrome andmultiple sclerosis in a subject and comprises administering apharmaceutically effective amount of a compound combination withanother, such as, for example, aspirin.

The present invention is not limited to the administration offormulations comprising a single compound of the invention. It is withinthe scope of the invention to provide combination treatments wherein aformulation is administered to a patient in need thereof that comprisesmore than one compound of the invention.

Conditions mediated by TNF-alpha include, but are not limited rheumatoidarthritis, Crohn's disease, ulcerative colitis, inflammatory bowelsyndrome and multiple sclerosis.

A compound useful in the present invention can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient or a domestic animal in a variety of forms adapted tothe chosen route of administration, i.e., orally or parenterally, byintranassally by inhalation, intravenous, intramuscular, topical orsubcutaneous routes.

A compound of the present invention can also be administered using genetherapy methods of delivery. See, e.g., U.S. Pat. No. 5,399,346, whichis incorporated by reference in its entirety. Using a gene therapymethod of delivery, primary cells transfected with the gene for thecompound of the present invention can additionally be transfected withtissue specific promoters to target specific organs, tissue, grafts,tumors, or cells.

Thus, the present compound may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compound may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the present compound can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compound to the skin are known to the art; for example, seeJacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S.Pat. No. 4,820,508).

Useful dosages of the compound can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compound(s) in a liquid composition,such as a lotion, will be from about 0.1-25 wt-%, preferably from about0.5-10 wt-%. The concentration in a semi-solid or solid composition suchas a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5wt-%.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician. Also the dosage of the compound varies depending on thetarget cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

The invention provides for an agent that is a modulator ofTNF-alpha/TNF-alpha-receptor interactions.

The candidate agent may be a synthetic agent, or a mixture of agents, ormay be a natural product (e.g. a plant extract or culture supernatant).A candidate agent according to the invention includes a small moleculethat can be synthesized, a natural extract, peptides, proteins,carbohydrates, lipids etc.

Candidate modulator agents from large libraries of synthetic or naturalagents can be screened. Numerous means are currently used for random anddirected synthesis of saccharide, peptide, and nucleic acid basedagents. Synthetic agent libraries are commercially available from anumber of companies including Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates(Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemicallibrary is available from Aldrich (Milwaukee, Wis.). Combinatoriallibraries are available and can be prepared. Alternatively, libraries ofnatural agents in the form of bacterial, fungal, plant and animalextracts are available from e.g., Pan Laboratories (Bothell, Wash.) orMycoSearch (NC), or are readily producible by methods well known in theart. Additionally, natural and synthetically produced libraries andagents are readily modified through conventional chemical, physical, andbiochemical means.

Useful agents may be found within numerous chemical classes. Usefulagents may be organic agents, or small organic agents. Small organicagents have a molecular weight of more than 50 yet less than about 2,500daltons, preferably less than about 750, more preferably less than about350 daltons. Exemplary classes include heterocycles, peptides,saccharides, steroids, and the like. The agents may be modified toenhance efficacy, stability, pharmaceutical compatibility, and the like.Structural identification of an agent may be used to identify, generate,or screen additional agents. For example, where peptide agents areidentified, they may be modified in a variety of ways to enhance theirstability, such as using an unnatural amino acid, such as a D-aminoacid, particularly D-alanine, by functionalizing the amino or carboxylicterminus, e.g. for the amino group, acylation or alkylation, and for thecarboxyl group, esterification or amidification, or the like.

For primary screening, a useful concentration of a candidate agentaccording to the invention is from about 10 mM to about 100 μM or more(i.e. 1 mM, 10 mM, 100 mM, 1 M etc.). The primary screeningconcentration will be used as an upper limit, along with nine additionalconcentrations, wherein the additional concentrations are determined byreducing the primary screening concentration at half-log intervals (e.g.for 9 more concentrations) for secondary screens or for generatingconcentration curves.

A high throughput screening kit according to the invention comprises allthe necessary means and media for performing the detection of an agentthat modulates TNF-alpha/TNF-alpha receptor interactions by interactingwith TNF-alpha in the presence of a polypeptide, preferably at aconcentration in the range of 1 μM to 1 mM.

The kit comprises the following. Recombinant cells of the invention,comprising and expressing the nucleotide sequence encoding TNF-alpha,which are grown according to the kit on a solid support, such as amicrotiter plate, more preferably a 96 well microtiter plate, accordingto methods well known to the person skilled in the art especially asdescribed in WO 00/02045. Alternatively TNF-alpha is supplied in apurified form to be immobilized on, for example, a 96 well microtiterplate by the person skilled in the art. Alternatively TNF-alpha issupplied in the kit pre-immobilized on, for example, a 96 wellmicrotiter plate. The TNF-alpha may be whole TNF-alpha or a fragmentthereof.

Modulator agents according to the invention, at concentrations fromabout 1 μM to 1 mM or more, are added to defined wells in the presenceof an appropriate concentration of anti-TNF-alpha polypeptide, anhomologous sequence thereof, a functional portion thereof or afunctional portion of an homologous sequence thereof, said concentrationof said polypeptide preferably in the range of 1 μM to 1 mM. Kits maycontain one or more anti-TNF-alpha polypeptides of the invention.

Binding assays are performed as according to the methods alreadydisclosed herein and the results are compared to the baseline level of,for example TNF-alpha binding to an anti-TNF-alpha polypeptide, anhomologous sequence thereof, a functional portion thereof or afunctional portion of an homologous sequence thereof, but in the absenceof added modulator agent. Wells showing at least 2 fold, preferably 5fold, more preferably 10 fold and most preferably a 100 fold or moreincrease or decrease in TNF-alpha-polypeptide binding (for example) ascompared to the level of activity in the absence of modulator, areselected for further analysis.

The invention provides for other kits useful for screening formodulators of TNF-alpha/TNF-alpha receptor binding, as well as kitsuseful for diagnosis of disorders characterised by dysfunction ofTNF-alpha. The invention also provides for kits useful for screening formodulators of disorders as well as kits for their diagnosis, saiddisorders characterised by one or more process involving TNF-alpha. Kitsuseful according to the invention can include an isolated TNF-alpha.Alternatively, or in addition, a kit can comprise cells transformed toexpress TNF-alpha. In a further embodiment, a kit according to theinvention can comprise a polynucleotide encoding TNF-alpha. In a stillfurther embodiment, a kit according to the invention may comprise thespecific primers useful for amplification of TNF-alpha. Kits usefulaccording to the invention can comprise an isolated TNF-alphapolypeptide, a homologue thereof, or a functional portion thereof. A kitaccording to the invention can comprise cells transformed to expresssaid polypeptide. Kits may contain more than one polypeptide. In afurther embodiment, a kit according to the invention can comprise apolynucleotide encoding TNF-alpha. In a still further embodiment, a kitaccording to the invention may comprise the specific primers useful foramplification of a macromolecule such as, for example, TNF-alpha. Allkits according to the invention will comprise the stated items orcombinations of items and packaging materials therefore. Kits will alsoinclude instructions for use.

Furthermore, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNanobodies of the invention onto other “scaffolds”, including but notlimited to human scaffolds or non-immunoglobulin scaffolds. Suitablescaffolds and techniques for such CDR grafting will be clear to theskilled person and are well known in the art, see for example U.S. Pat.No. 7,180,370, WO 01/27160, EP 0 605 522, EP 0 460 167, U.S. Pat. No.7,054,297, Nicaise et al., Protein Science (2004), 13:1882-1891; Ewertet al., Methods, 2004 October; 34(2):184-199; Kettleborough et al.,Protein Eng. 1991 October; 4(7): 773-783; O'Brien and Jones, MethodsMol. Biol. 2003: 207: 81-100; and Skerra, J. Mol. Recognit. 2000: 13:167-187, and Saerens et al., J. Mol. Biol. 2005 Sep. 23; 352(3):597-607,and the further references cited therein. For example, techniques knownper se for grafting mouse or rat CDR's onto human frameworks andscaffolds can be used in an analogous manner to provide chimericproteins comprising one or more of the CDR's of the Nanobodies of theinvention and one or human framework regions or sequences.

Thus, in another embodiment, the invention comprises a chimericpolypeptide comprising at least one CDR sequence chosen from the groupconsisting of CDR1 sequences, CDR2 sequences and CDR3 sequencesmentioned herein for the Nanobodies of the invention. Preferably, such achimeric polypeptide comprises at least one CDR sequence chosen from thegroup consisting of the CDR3 sequences mentioned herein for theNanobodies of the invention, and optionally also at least one CDRsequence chosen from the group consisting of the CDR1 sequences and CDR2sequences mentioned herein for the Nanobodies of the invention. Forexample, such a chimeric polypeptide may comprise one CDR sequencechosen from the group consisting of the CDR3 sequences mentioned hereinfor the Nanobodies of the invention, one CDR sequence chosen from thegroup consisting of the CDR1 sequences mentioned herein for theNanobodies of the invention and one CDR sequence chosen from the groupconsisting of the CDR1 sequences and CDR2 sequences mentioned herein forthe Nanobodies of the invention. The combinations of CDR's that arementioned herein as being preferred for the Nanobodies of the inventionwill usually also be preferred for these chimeric polypeptides.

In said chimeric polypeptides, the CDR's may be linked to further aminoacid sequences and/or may be linked to each other via amino acidsequences, in which said amino acid sequences are preferably frameworksequences or are amino acid sequences that act as framework sequences,or together form a scaffold for presenting the CDR's. Reference is againmade to the prior art mentioned in the last paragraph. According to onepreferred embodiment, the amino acid sequences are human frameworksequences, for example V_(H)3 framework sequences. However, non-human,synthetic, semi-synthetic or non-immunoglobulin framework sequences mayalso be used. Preferably, the framework sequences used are such that (1)the chimeric polypeptide is capable of binding TNF-alpha, i.e. with anaffinity that is at least 1%, preferably at least 5%, more preferably atleast 10%, such as at least 25% and up to 50% or 90% or more of theaffinity of the corresponding Nanobody of the invention; (2) thechimeric polypeptide is suitable for pharmaceutical use; and (3) thechimeric polypeptide is preferably essentially non-immunogenic under theintended conditions for pharmaceutical use (i.e. indication, mode ofadministration, doses and treatment regimen) thereof (which may beessentially analogous to the conditions described herein for the use ofthe Nanobodies of the invention).

According to one non-limiting embodiment, the chimeric polypeptidecomprises at least two CDR sequences (as mentioned above) linked via atleast one framework sequence, in which preferably at least one of thetwo CDR sequences is a CDR3 sequence, with the other CDR sequence beinga CDR1 or CDR2 sequence. According to a preferred, but non-limitingembodiment, the chimeric polypeptide comprises at least two CDRsequences (as mentioned above) linked at least two framework sequences,in which preferably at least one of the three CDR sequences is a CDR3sequence, with the other two CDR sequences being CDR1 or CDR2 sequences,and preferably being one CDR1 sequence and one CDR2 sequence. Accordingto one specifically preferred, but non-limiting embodiment, the chimericpolypeptides have the structure FR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4′, inwhich CDR1, CDR2 and CDR3 are as defined herein for the CDR's of theNanobodies of the invention, and FR1′, FR2′, FR3′ and FR4′ are frameworksequences. FR1′, FR2′, FR3′ and FR4′ may in particular be Framework 1,Framework 2, Framework 3 and Framework 4 sequences, respectively, of ahuman antibody (such as V_(H)3 sequences) and/or parts or fragments ofsuch Framework sequences. It is also possible to use parts or fragmentsof a chimeric polypeptide with the structureFR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4. Preferably, such parts or fragmentsare such that they meet the criteria set out in the preceding paragraph.

The invention also relates to proteins and polypeptides comprisingand/or essentially consisting of such chimeric polypeptides, to nucleicacids encoding such proteins or polypeptides; to methods for preparingsuch proteins and polypeptides; to host cells expressing or capable ofexpressing such proteins or polypeptides; to compositions, and inparticular to pharmaceutical compositions, that comprise such proteinsor polypeptides, nucleic acids or host cells; and to uses of suchproteins or polypeptides, such nucleic acids, such host cells and/orsuch compositions, in particular for prophylactic, therapeutic ordiagnostic purposes, such as the prophylactic, therapeutic or diagnosticpurposes mentioned herein. For example, such proteins, polypeptides,nucleic acids, methods, host cells, compositions and uses may beanalogous to the proteins, polypeptides, nucleic acids, methods, hostcells, compositions and use described herein for the Nanobodies of theinvention.

It should also be noted that, when the Nanobodies of the inventionscontain one or more other CDR sequences than the preferred CDR sequencesmentioned above, these CDR sequences can be any suitable (i.e. suitablefor the purposes described herein) CDR sequences and/or these CDRsequences can be obtained in any manner known per se, for example fromNanobodies (preferred), V_(H) domains from conventional antibodies (andin particular from human antibodies), heavy chain antibodies,conventional 4-chain antibodies (such as conventional human 4-chainantibodies) or other immunoglobulin sequences directed against TNF. Suchimmunoglobulin sequences directed against xxxx can be generated in anymanner known per se, as will be clear to the skilled person, i.e. byimmunization with TNF or by screening a suitable library ofimmunoglobulin sequences with TNF, or any suitable combination thereof.Optionally, this may be followed by techniques such as random orsite-directed mutagenesis and/or other techniques for affinitymaturation known per se. Suitable techniques for generating suchimmunoglobulin sequences will be clear to the skilled person, and forexample include the screening techniques reviewed by Hoogenboom, NatureBiotechnology, 23, 9, 1105-1116 (2005). Other techniques for generatingimmunoglobulins against a specified target include for example theNanoclone technology (as for example described in the non-prepublishedU.S. provisional patent application 60/648,922), so-called SLAMtechnology (as for example described in the European patent application0 542 810), the use of transgenic mice expressing human immunoglobulinsor the well-known hybridoma techniques (see for example Larrick et al,Biotechnology, Vol. 7, 1989, p. 934). All these techniques can be usedto generate immunoglobulins against TNF, and the CDR's of suchimmunoglobulins can be used in the Nanobodies of the invention, i.e. asoutlined above. For example, the sequence of such a CDR can bedetermined, synthesized and/or isolated, and inserted into the sequenceof a Nanobody of the invention (e.g. so as to replace the correspondingnative CDR), all using techniques known per se such as those describedherein, or Nanobodies of the invention containing such CDR's (or nucleicacids encoding the same) can be synthesized de novo, again using thetechniques mentioned herein.

The invention will now be further described by means of the followingnon-limiting examples and figures, in which the Figures show:

MONOVALENT TNFα NANOBODIES

FIG. 1: Sequence alignment of human TNFα nanobodies

FIG. 2: Sequence alignment of serum albumin specific TNFα nanobodies

FIG. 3: Binding of albumin specific TNFα nanobodies to human serumalbumin

FIG. 4: Binding of albumin specific TNFα nanobodies to rhesus serumalbumin

FIG. 5: Binding of albumin specific TNFα nanobodies to mouse serumalbumin

FIG. 6: Purity of TNFα and serum albumin nanobodies (SDS-PAGE)

FIG. 7: Western Blot analysis of TNFα and serum albumin nanobodies

FIG. 8: Binding of TNFα nanobodies to human TNFα (ELISA)

FIG. 9: Binding of TNFα nanobodies to rhesus TNFα (ELISA)

FIG. 10: Receptor-inhibition assay on Enbrel for human TNFα

FIG. 11: Receptor-inhibition assay on Enbrel for rhesus TNFα

FIG. 12: Binding of TNFα nanobodies to human TNFα (Biacore)

FIG. 13: Binding of TNFα nanobodies to rhesus TNFα (Biacore)

FIG. 14: Binding of TNFα nanobodies to Protein A (Biacore)

FIG. 15: Temperature treatment of TNFα and serum albumin nanobodies(Western Blot)

FIG. 16: Stability: temperature treatment of TNFα nanobodies (ELISA)

FIG. 17: Temperature treatment of serum albumin nanobodies (Biacore)

Bivalent TNFα nanobodies

FIG. 18: Purity of bivalent TNFα nanobodies (SDS-PAGE)

FIG. 19: Western Blot analysis of bivalent TNFα nanobodies

FIG. 20: Receptor-inhibition assay on Enbrel for bivalent TNFαnanobodies

FIG. 21: Stability: temperature treatment of bivalent TNFα nanobodies(ELISA)

Humanised monovalent TNFα nanobodies

FIG. 22: Multiple sequence alignment of TNF1 humanised nanobodies; DP51is SEQ ID NO: 472, DP53 is SEQ ID NO: 473.

FIG. 23: Multiple sequence alignment of TNF2 humanised nanobodies; DP54is SEQ ID NO: 474.

FIG. 24: Multiple sequence alignment of TNF3 humanised nanobodies; DP29is SEQ ID NO: 475.

FIG. 25: Multiple sequence alignment of ALB1 humanised nanobodies

FIG. 26: Purity of humanised TNFα and serum albumin nanobodies(SDS-PAGE)

FIG. 27: Western Blot analysis of humanised TNFα and serum albuminnanobodies

FIG. 28: Binding of humanised TNFα nanobodies to human TNFα

FIG. 29: Binding of humanised serum albumin nanobodies to human serumalbumin

FIG. 30: Stability: temperature treatment of humanised TNFα nanobodies(ELISA)

Trivalent TNFα nanobodies

FIG. 31: Purity of trivalent TNFα nanobodies (SDS-PAGE)

FIG. 32: Western Blot analysis of trivalent TNFα nanobodies

FIG. 33: Stability: temperature treatment of trivalent TNFα nanobodies(ELISA)

Humanised monovalent TNFα nanobodies (second round)

FIG. 34: Multiple sequence alignment of TNF1 humanised nanobodies

FIG. 35: Multiple sequence alignment of TNF2 humanised nanobodies

FIG. 36: Multiple sequence alignment of TNF3 humanised nanobodies

FIG. 37: Multiple sequence alignment of ALB1 humanised nanobodies

FIG. 38: Purity of humanised TNFα nanobodies (SDS-PAGE)

FIG. 39: Western Blot analysis of humanised TNFα nanobodies

FIG. 40: Binding of humanised TNFα nanobodies to human TNFα

FIG. 41: Stability: temperature treatment of humanised TNFα nanobodies(ELISA)

FIG. 42: Analysis of purified TNF60 on Silver stained SDS-PAGE gel (A)Coomassie stained SDS-PAGE gel (B) and in Western blot analysis usinganti-NB (C) for detection

FIG. 43: Chromatogram of analytical size exclusion of TNF60 on SuperdexHR75

FIG. 44: Binding of TNF60 to human TNF-alpha

FIG. 45: Dose response curve obtained in cytotoxicity assay with humanTNF-alpha using Nanobody™ TNF60 in comparison with Enbrel (Etanercept),Humira (Adalimumab) and Remicade (Infliximab)

FIG. 46: Dose response curve obtained in cytotoxicity assay with rhesusTNFα using Nanobody™ TNF60 in comparison with Enbrel (Etanercept),Humira (Adalimumab) and Remicade (Infliximab)

FIG. 47: Pharmacokinetic profile of TNF60 in mice

FIG. 48: Immunogenicity profile of TNF60 in mice

FIG. 49: Analysis of purified TNF56-PEG40, TNF56-PEG60, TNF56-biotine,TNF55-PEG40, TNF55-PEG60 and TNF55-biotine on Coomassie stained SDS-PAGEgel FIG. 50: Analysis of purified TNF56-PEG40 on SDS-PAGE gel usingSilver stain (A) Coomassie stain (B) and in Western blot analysis usinganti-NB (C) for detection

FIG. 51: Chromatogram of analytical size exclusion of TNF56-PEG40 onSuperdex HR 75

FIG. 52: Chromatogram of analytical size exclusion of TNF56-PEG40 onSuperdex HR 200

FIG. 53: Dose response curve obtained in cytotoxicity assay with humanTNFα using Nanobody™ TNF56-PEG40 and the monovalent wild-type Nanobody™TNF1 in comparison with Enbrel (Etanercept), Humira (Adalimumab) andRemicade (Infliximab)

FIG. 54: Dose response curve obtained in cytotoxicity assay with rhesusTNFα using Nanobody™ TNF56-PEG40 in comparison with Enbrel (Etanercept),Humira (Adalimumab) and Remicade (Infliximab)

FIG. 55: Pharmacokinetic analysis of pegylated bivalent Nanobody™TNF56-PEG40 and TNF56-PEG60 after intravenous administration in mice

FIG. 56: Pharmacokinetic analysis of pegylated bivalent Nanobody™3E-3E-PEG20, pegylated bivalent Nanobody™ 3E-3E-PEG40 and bispecificNanobody™ 3E-3E-AR1 after intravenous administration in mice

FIG. 57: Immunogenicity profile of TNF56-PEG40 and TNF56-PEG60 in mice

FIG. 58: Efficacy of TNF60 in the prevention of chronic polyarthritis inmice

FIG. 59: Efficacy of TNF60 in therapeutic treatment of chronicpolyarthritis in mice

FIG. 60: Effect of TNF60 Nanobody™ formatting on efficacy in theprevention of chronic polyarthritis in mice

FIG. 61: Sequence alignment of Nanobodies™ PMP1C2, 3E, 1A and 3G

FIG. 62: Molecular model of TNF-60

The appended Tables form an integral part of the present specificationand are as follows:

Monovalent TNFα nanobodies

Table 8: Sequence listing of TNFα nanobodies

Table 9: Koff values of human TNFα nanobodies

Table 10: Homology of TNFα and serum albumin nanobodies to humangermline sequences

Table 11: Expression levels of TNFα and serum albumin nanobodies

Table 12: ELISA binding to human and rhesus TNFα

Table 13: Receptor-inhibition assay of TNFα nanobodies

Table 14: Biacore analysis of TNFα nanobodies

Table 15: Binding of TNFα nanobodies to TNFα (K_(D)-values)

Table 16: Potency of TNFα nanobodies to neutralize human (a) and rhesus(b) TNFα

Table 17: OD280 nm of TNFα and serum albumin nanobodies aftertemperature treatment

Table 18: Potency of TNFα nanobodies after temperature treatment

Bivalent TNFα nanobodies

Table 19: Sequence listing of bivalent TNFα nanobodies and linkersequences

Table 20: Bivalent TNFα nanobody constructs

Table 21: Expression levels of bivalent TNFα nanobodies

Table 22: Receptor-inhibition assay of bivalent TNFα nanobodies

Table 23: Potency of TNFα nanobodies to neutralize human (a) and rhesus(b) TNFα

Table 24: OD280 nm of bivalent TNFα nanobodies

Humanised monovalent TNFα nanobodies

Table 25: Sequence listing of humanised monovalent TNFα and serumalbumin nanobodies

Table 26: Expression levels of humanised TNFα and serum albuminnanobodies

Table 27: Potency of TNFα nanobodies to neutralize human TNFα

Table 28: OD280 nm of humanised TNFα and serum albumin nanobodies

Trivalent TNFα nanobodies

Table 29: Sequence listing of trivalent TNFα nanobodies

Table 30: Trivalent TNFα nanobody constructs

Table 31: Expression levels of trivalent TNFα nanobodies

Table 32: Potency of trivalent TNFα nanobodies to neutralize human TNFα

Table 33: Binding of trivalent nanobodies to serum albumin(K_(D)-values)

Table 34: OD280 nm of trivalent TNFα nanobodies

Humanised monovalent TNFα nanobodies (second round)

Table 35: Sequence listing of second round humanised monovalent TNFαnanobodies

Table 36: Expression levels of humanised TNFα nanobodies

Table 37: Potency of TNFα nanobodies to neutralize human TNFα

Table 38: OD280 nm of humanised TNFα nanobodies

Table 39: Comparing bio-activity of nanobodies

Further tables

Table 40: Overview of oligonucleotides used in formatting of trivalentNanobodies™

Table 41: Overview of oligonucleotides used in cloning of trivalentNanobodies™

Table 42: EC50 values obtained in cytotoxicity assay using trivalentNanobody™ TNF60 in comparison with commercial controls (Enbrel,Remicade, Humira)

Table 43: Affinity determination of TNF60 and TNF24 on human serumalbumin in Biacore. Nd, not determined.

Table 44: Overview of oligonucleotides used in formatting of bivalentNanobodies™

Table 45: EC50 values obtained in cytotoxicity assay using bivalentNanobodies™ in comparison with commercial controls (Enbrel, Remicade,Humira)

Table 46: Results of synovium derived fibroblast studies

Table 47: Results of murine air pouch studies

EXAMPLES Example 1: Identification of TNFα and Serum Albumin SpecificNanobodies

Antagonistic nanobodies were identified using two llamas (Llama glama)immunized with human TNFα by 6 injections of 100 μg of the cytokine atweekly intervals. Screening was performed using a competition basedassay, in which individual nanobodies were analyzed for their capabilityto inhibit binding of labeled TNFα to its receptor. The albumin specificnanobodies were identified from a llama immunized with human serumalbumin. Screening of individual nanobodies was performed by ELISA usinghuman, rhesus and mouse albumin, yielding a panel of nanobodiescross-reacting with the serum albumin of various species.

Example 2: Sequence Analysis of Isolated Nanobodies

Different classes of nanobodies were identified based on sequenceanalysis (FIG. 1) using a BLOSUM62 scoring matrix and a similaritysignificance value cut-off of ≥60%: Class I (PMP1 C2, PMP1 G11, PMP1H6), Class II (PMP1 G5, PMP1 H2, PMP3 G2), Class IIb (PMP1 D2), ClassIII (PMP3 D10, PMP5 F10). Table 8 lists the sequences of these TNFαnanobodies (SEQ ID NOs: 52 to 60).

Based on sequence analysis (FIG. 2) different classes of serum albuminnanobodies were identified using the BLOSUM62 scoring matrix and asimilarity significance value cut-off of ≥60%. Table 8 lists thesequences of these serum albumin nanobodies (SEQ ID NOs: 61 to 67).

Example 3: Biacore Analysis

TNFα

Binding of nanobodies to TNFα was characterised by surface plasmonresonance in a Biacore 3000 instrument. TNF from different species wascovalently bound to CMS sensor chips surface via amine coupling until anincrease of 250 response units was reached. Remaining reactive groupswere inactivated. Nanobody binding was assessed at one concentration (1in 1,000 diluted). Each nanobody was injected for 4 minutes at a flowrate of 45 μl/min to allow for binding to chip-bound antigen. Bindingbuffer without nanobody was sent over the chip at the same flow rate toallow spontaneous dissociation of bound nanobody for 4 hours.K_(off)-values were calculated from the sensorgrams obtained for thedifferent nanobodies.

Of each class of nanobodies unpurified proteins were analyzed inBiacore. K_(off) data is listed in Table 9.

Representative nanobodies from each class were retained for furtheranalysis based on k_(off) value. For Class I PMP1C2 (TNF1) was selected;PMP1G5 (TNF2) was selected as representative of Class II; PMP5F10 (TNF3)was selected as representative of Class III.

Serum Albumin

Binding was assayed as described above except that 1 in 20 dilutionswere used. FIGS. 3, 4 and 5 illustrate screening of albumin specificTNFα nanobodies versus human, rhesus and mouse serum albumin usingunpurified protein.

The nanobodies are ranked according to k_(off)-values, see Table IIIbelow:

TABLE III Class Human Rhesus Mouse C PMP6A8 PMP6A8 PMP6B4 C PMP6B4PMP6B4 PMP6A8 B PMP6A6 PMP6A6 PMP6A6 B PMP6C1 PMP6C1 PMP6C1 A PMP6G8PMP6G8 PMP6G8 A PMP6A5 PMP6A5 PMP6A5 D PMP6G7 PMP6G7 PMP6G7

The best k_(off) were obtained for members of family C and family B.Cross-reactivity between mouse, human and rhesus serum albumin was alsoobserved for members of those families. A representative nanobody fromclass B and C was defined for further analysis: PMP6A6 (ALB1) wasselected as representative of Class B and PMP6A8 (ALB2) was selected asrepresentative of Class C.

Example 4: Cloning of Monovalent Nanobodies in pAX051

Description of Escherichia coli Expression Vector

pAX051 is a derivative of pUC19. It contains the LacZ promoter whichenables a controlled induction of expression using IPTG. The vector hasa resistance gene for Ampicillin or Carbenicillin. The multicloningsites harbours several restriction sites of which SfiI and BstEII arefrequently used for cloning of Nanobodies™. In frame with the NB codingsequence the vector codes for a C-terminal c-myc tag and a (His)6 tag.The signal peptide is the gen3 leader sequence which translocates theexpressed Nanobody™ to the periplasm.

The DNA coding for the selected nanobodies TNF1 (PMP1C2), TNF2 (PMP1G5),TNF3 (PMP5F10), ALB1 (PMP6A6) and ALB2 (PMP6A8) was cloned in pAX051 andthe construct was transformed to TG1 electrocompetent cells. Clones wereanalyzed for PCR insert and the nucleotide sequence was determined from4 positive clones. Glycerol stocks were prepared from clones containingthe correct sequence and stored at −80° C.

Example 5: Expression of Monovalent Nanobodies

A preculture was started by inoculating a single colony of the cloneexpressing the respective nanobodies at 37° C. in Luria Broth,Ampicillin/Carbenicillin (100 μg/ml) and 2% glucose overnight. Thispreculture was used to inoculate. Inoculum is 1% percent (v/v) of theproduction culture (TB medium+Ampicillin/Carbenicillin+0.1% Glucose).The production culture is grown at 37° C. until an OD600 nm of 5-10 isreached and nanobody expression is induced by adding IPTG (1 mM finalconcentration). Protein expression is allowed to continue either for 4 hat 37° C. or overnight at 28° C., at which point cells are collected bycentrifugation and stored as wet cell paste at −20° C.

Preparative periplasmic extracts of the −20° C. stored wet cell pasteare made by resuspending the pellet in Peri-buffer (50 mM NaH₂PO₄, 300mM NaCl, adjusted pH to 8.0), rotating the mixture for 30 min at 4° C.and centrifuging the mixture using a preparative centrifuge (SorvallRC-3C Plus with H-6000A rotor) to pellet the cells. Supernatant,representing a rough extract of the periplasmic space, is collected forfurther purification.

The His(6)-tagged nanobodies are purified on Immobilized Metal AffinityChromatography (IMAC). The TALON resin (Clontech) is processed accordingto the manufacturer's instructions. The extracts are incubated with theresin for 30 min at RT on a rotator. The resin is washed with PBS andtransferred to a column. The packed resin is washed with 15 mMImidazole. The nanobodies are eluted from the column using 150 mMImidazole. The eluated fractions are analyzed by spotting on HybondMembrane and visualization with Ponceau. Fractions containing proteinare pooled and dialysed against PBS. Dialysed proteins are collected,filter sterilized, concentration determined and stored in aliquots at−20° C.

Characterisation of Monovalent TNFα Nanobodies

Example 6: Homology to Human Germline Sequences

The nanobody amino acid sequences were compared to the human germlinesequences as represented in Table 10. In order of homology to humansequences the nanobodies rank as follows: TNF1>TNF2>TNF3 for the TNFαnanobodies; ALB1>ALB2 for the serum albumin nanobodies.

Example 7: Expression Level

Expression levels were calculated and represented in Table 11. In orderof yield the nanobodies rank as follows: TNF1>TNF2>TNF3 for the TNFαnanobodies; ALB1>ALB2 for the serum albumin nanobodies.

Example 8: SDS-Page Analysis

To determine the purity, protein samples were analyzed on a 15% SDS-PAGEgel. 10 μl Laemmli sample buffer was added to 10 μl (1 ug) purifiedprotein, the sample was heated for 10 minutes at 95° C., cooled andloaded on a 15% SDS-PAGE gel. The gel was processed according to generalprocedures and stained with Coomassie Brilliant Blue (CBB). FIG. 6represents the SDS-PAGE for the TNFα-specific and serum albumin-specificnanobodies.

Example 9: Western Blot Analysis

100 ng of purified protein was loaded on the gel. Following SDS-PAGE,proteins were transferred to a nitrocellulose membrane using the MiniTrans-Blot® Electrophoretic Transfer Cell (Biorad). The membrane wasblocked overnight in PBS, 1% casein at 4° C. As all constructs werefused to c-myc tag, mouse monoclonal anti-myc antibody was used as adetection tool. In addition, rabbit polyclonal anti-Nanobody (R23) wasused as a detection tool. The blot was incubated for 1 h at roomtemperature with agitation in 1/2000 diluted anti-myc antibody in PBS or1/2000 anti-Nanobody antibody in PBS, 1% casein. The membrane was washed5 times in PBS before the secondary antibody was applied(rabbit-anti-mouse IgG alkaline phosphatase conjugate, Sigma, A1902,diluted 1/1000 in PBS or goat anti-rabbit IgG alkaline phosphataseconjugate, Sigma, A8025, 1% casein). After incubation with gentleagitation for 1 h at room temperature, the membrane was washed 5 timesin PBS. Blots were developed using BCIP/NBT solutions and the reactionwas stopped by washing the blot with milliQ water when bands wereclearly visible. FIG. 7 represents the Western Blot analysis.

Example 10: ELISA Binding to Human and Rhesus TNFα

An ELISA was performed to examine binding to human and rhesus TNFα. A96-well Maxisorp plate was coated with 2 μg/ml Neutravidin in PBS ON at4° C. Plates were blocked with 1% caseine for 2 hrs at RT. BiotinylatedTNFα (400 ng/ml) was added to the wells and incubated for 1 hr at RT.Nanobody samples were diluted starting at 2 μg/ml and using 1 in 3dilutions. Nanobodies were detected using mouse anti-myc (1/2000diluted) and rabbit anti-mouse alkaline phosphatase (1/2000 diluted,Sigma, A1902) and pNPP (2 mg/ml) as substrate. FIGS. 9 and 10 representthe binding in ELISA to human and rhesus TNFα.

Results are summarized in Table 12. TNF1 and TNF3 show binding to bothhuman and rhesus TNFα. TNF2 is binding to human TNFα but is only weaklyreactive to rhesus TNFα.

Example 11: Receptor-Inhibition Assay

The ability to inhibit receptor-ligand interaction was analyzed forrhesus and human TNFα. A 96-well Maxisorp plate was coated with 2 μg/mlEnbrel in PBS ON at 4° C. Plates were blocked with 1% Caseine for 2 hrsat RT. Nanobody samples were pre-incubated for 30 min at RT withbiotinylated TNFα (10 ng/ml) starting at a concentration of 5 μg/ml andusing 1 in 2 dilutions. Samples were added to the plates and incubatedfor 1 hr at RT. Biotinylated TNFα was detected using Extravidin alkalinephosphatase (1/2000 diluted) and pNPP (2 mg/ml) as substrate. FIGS. 11and 12 represent an inhibition ELISA for human and rhesus TNFα. Resultsare summarized in Table 13. Inhibition of ligand/receptor binding isobserved for TNF1 and TNF3 for both human and rhesus TNF, while TNF2 isonly inhibiting human TNFα.

Example 12: Biacore Analysis

TNFα Binding

The analysis was performed as described in Example 3. FIGS. 13 and 14illustrate the binding to human and rhesus TNFα via Biacore analysis.Results are summarized in Table 14. Binding experiments in Biacoreconfirm the ELISA results: cross-reactive binding for TNF1 and TNF3,while TNF2 only significantly binds human TNFα.

Serum Albumin

Binding was assayed as described above except that series of differentconcentrations were used. Each concentration was injected for 4 minutesat a flow rate of 45 μl/min to allow for binding to chip-bound antigen.Binding buffer without analyte was sent over the chip at the same flowrate to allow for dissociation of bound nanobody. After 15 minutes,remaining bound analyte was removed by injection of the regenerationsolution (25 mM NaOH).

From the sensorgrams obtained for the different concentrations of eachanalyte K_(D)-values were calculated via steady state affinity whenequilibrium was reached.

Results are summarized in Table 15. Cross-reactivity is observed forboth ALB1 and ALB2. The highest affinity is observed for ALB2 on humanand rhesus TNFα. However, the difference in affinity for human/rhesusversus mouse serum albumin is more pronounced for ALB2 (factor 400),while for ALB1 a difference of a factor 12 is observed.

Example 13: Bio-Assay

The TNFα sensitive mouse fibroblast cell line L929s was used formeasuring the anti-TNFα activity of the selected nanobodies. At asufficiently high concentration of TNFα in the medium, i.e. cytotoxicdose, L929s cells undergo necrosis. The inhibition of TNFα interactionwith its receptor was determined by pre-incubating a series of antibodydilutions with a cytotoxic concentration of TNFα before adding themixture to the cells. The presence of actinomycin D in the mediumsensitises the cells further to TNFα, resulting in increased sensitivityof the bioassay for free TNFα.

The L929 cells were grown to nearly confluency, plated out in 96-wellmicrotiter plates at 5000 cells per well and incubated overnight.Actinomycin D was added to the cells at a final concentration of 1μg/ml. Serial dilutions of the nanobodies to be tested were mixed with acytotoxic concentration of TNFα (final assay concentration is 0.5 ng/mlor 15 IU/ml). After at least 30 minutes of incubation at 37° C., thismixture was added to the plated cells. Plates were incubated for 24hours at 37° C. and 5% CO₂. Cell viability was determined by use of thetetrazolium salt WST-1. Dose-response curves and EC₅₀ values werecalculated with Graphpad Prism.

The results are summarized in Table 16 for human and rhesus TNFα. Basedon their potency to neutralize cytotoxic activity, the molecules areranked as follows: TNF3>TNF1>TNF2 for human TNFα, and TNF1=TNF3>TNF2 forrhesus TNFα.

Example 14: Protein a Binding

FIG. 14 represents Protein A binding analyzed in Biacore as described inExample 12. Positive binding was obtained for TNF1, TNF2, ALB1. No orweak binding was observed for TNF3 and ALB2.

Example 15: Temperature Stability

Samples were diluted at 200 μg/ml and divided in 8 aliquots containing500 μl. The different vials were incubated each at a given temperatureranging from RT to 90° C. After treatment the samples were cooled downfor 2 hrs at RT, they were kept at 4° C. Precipitates were removed bycentrifugation for 30 min at 14,000 rpm. SN was carefully removed andfurther analysed.

OD280 nm

OD at 280 nm was measured and the concentration was calculated. Resultsare summarized in Table 17. A decrease in protein content was observedfor TNF2 and TNF3 starting at 80° C., while for ALB2 a decrease isobserved starting from 70° C.

Western Blot

2 μg of treated protein was separated on a 15% SDS-PAGE and transferredto a nitrocellulose membrane and treated as described above. Detectionwas performed using polyclonal anti-Nanobody (R23, 1/2000 diluted) andanti-rabbit horse radish peroxidase (DAKO, P0448, 1/2000 diluted). FIG.15 represents the Western Blot analysis. A clear drop in proteinconcentration was observed for ALB2 treated at 70, 80 and 90° C.Aggregation was still observed for TNF1 treated at 70, 80 and 90° C.;for TNF3 treated at 90° C.; for ALB1 treated at 90° C., meaning that theSN still contains traces of precipitates which result in a higher OD280nm read-out. This explains why the protein concentration as measured atOD280 nm does not decrease for TNF1, TNF3 and ALB1 treated at thesehigher T.

ELISA

The ELISA to detect binding to human TNFα was essentially performed asdescribed in Example 10. Results are presented in FIG. 16. Human TNFαbinding is decreased for TNF1, TNF2, TNF3 starting at 80° C.

Bio-Assay

The bio-assay was performed as described in Example 13. The results aresummarized in Table 18. Potency of the nanobodies is decreased for TNF1starting at 70° C.; for TNF2 and TNF3 starting at 80° C.

Biacore

Binding to human serum albumin was determined as described in Example12. A fixed concentration was used (1 in 50 diluted). Results arepresented in FIG. 17. Temperature treatment is not influencing bindingto serum albumin for ALB1. The treatment has an effect on the k_(on) forALB2 starting from T=70° C.

Bivalent Nanobodies

Example 16: Formatting of Bivalent TNFα Specific Nanobodies

TNF1, TNF2 and TNF3 were formatted to bivalent nanobodies. As spacerbetween the two building blocks either a 9AA GlySer linker (Table 19 SEQID No: 68) or a 30 AA GlySer linker (Table 19 SEQ ID No: 69) was used.This generated the constructs represented by Table 20. Table 19 liststhe sequences of these bivalent TNFα nanobodies (SEQ ID NOs: 70 to 75).

Example 17: Expression of Bivalent TNFα Specific Nanobodies

Expression was performed as described in Example 5. The His(6)-taggednanobodies were purified on Immobilized Metal Affinity Chromatography(IMAC). The Ni-NTA resin (Qiagen) was processed according to themanufacturer's instructions. The extracts were incubated with the resinand incubated for 30 min at RT on a rotator. The resin was washed withPBS and transferred to a column. The packed resin was washed with PBS (1in 10 diluted). The column was pre-eluted with 15 mM Imidazole. Thenanobodies were eluted from the column using 25 mM Citric Acid pH=4. Theeluated fractions were analyzed by spotting on Hybond Membrane and byvisualization with Ponceau. Fractions containing protein were pooled andfurther purified on Cation exchange followed by size exclusion. Purifiedproteins were collected, filter sterilized, concentration determined andstored in aliquots at −20° C.

Characterisation of Bivalent TNFα Specific Nanobodies

Example 18: Expression Level

Expression levels of the bivalent TNFα nanobodies were calculated andrepresented in Table 21. The linker has no significant effect on theexpression level of the nanobodies.

Example 19: SDS-PAGE

SDS-Page was performed as described in Example 8. FIG. 18 shows theresult of the SDS-Page.

Example 20: Western Blot

Western Blot analysis was performed as described in Example 9. FIG. 19represents the Western Blot results.

Example 21: Receptor-Inhibition Assay

The assay was performed as described in Example 11. FIG. 20 and Table 22represent the results. Enhancement of inhibition of ligand/receptorbinding was observed for all bivalent nanobodies compared to themonovalent format.

Example 22: Bio-Assay

The assay was performed as described in Example 13. Results aresummarized in Table 23. Based on their potency to neutralize cytotoxicactivity TNF8, TNF7, TNF9 and TNF5 have a potency in the range ofEnbrel.

Example 23: Temperature Stability

Samples were analysed as described in Example 15.

OD280 nm

OD at 280 nm was measured and the concentration was calculated. Resultsare summarized in Table 24. A decrease in protein content was observedfor TNF4 and TNF7 starting at 70° C., while for TNF5, TNF6, TNF8 andTNF9 a decrease was observed starting from 80° C.

Western Blot

Samples were analyzed for the presence of aggregates as described inExample 15.

ELISA

The ELISA to detect binding to human TNFα was essentially performed asdescribed above. Results are presented in FIG. 21. Human TNFα bindingwas decreased for TNF5, TNF6, TNF8 and TNF9 starting at 80° C., for TNF4and TNF7 starting from 70° C.

Humanised Monovalent Nanobodies

Example 24: Identification of Non-Human Amino Acid Positions in TNFα andSerum Albumin Specific Nanobodies

FIG. 22 (TNF1), FIG. 23 (TNF2), FIG. 24 (TNF3) and FIG. 25 (ALB1)represent multiple sequence alignments (Clustal W 1.7) with DP51, DP53,DP54 and DP29 sequences.

In addition to the amino acid mutations, codon optimization wasperformed yielding the sequences of Table 25 SEQ ID NOs: 76 to 89(Nanobodies against TNF-alpha and human serum albumin, respectively).

Example 25: Generation of Codon Optimised Mutants

Oligonucleotides were synthesised spanning the entire sequence of thenanobodies.

Example 26: Expression of Bivalent TNFα Specific Nanobodies

Expression was performed as described in Example 5.

Characterisation of Humanised Nanobody

Example 27: Expression Level

Table 26 represents calculated expression levels. Expression wasachieved with yields in the range of 3.5-11.7 mg/ml. Induction time didnot influence the yield.

Example 28: SDS-PAGE

SDS-PAGE was performed as described in Example 8. FIG. 26 represents theSDS-PAGE gel.

Example 29: Western Blot

Western Blot analysis was performed as described in Example 9. FIG. 27represents the Western Blot results.

Example 30: Bio-Assay

The assay was performed as described in Example 13.

The results of the humanised nanobodies are summarized in Table 27. Thewildtype nanobodies are included as reference.

Example 31: Biacore

The analysis was performed as described in Example 12. FIGS. 28 and 31shows Biacore results.

Example 32: Temperature Stability

Samples were analysed as described in Example 15.

OD280 nm

OD at 280 nm was measured and the concentration was calculated. Resultsare summarized in Table 28.

No significant decrease in protein concentration is observed for thehumanised TNF1 nanobodies (TNF13-14). A decrease in proteinconcentration is observed for humanised TNF2 (TNF15-19) and TNF3(TNF20-23) starting at 80° C. A decrease in protein concentration isobserved for humanized ALB1 (ALB4-5) starting at 70° C. and for ALB3starting at 60° C.

Western Blot

Samples were analyzed for the presence of aggregates as described inExample 15.

ELISA

The ELISA to detect binding to human TNFα was essentially performed asdescribed in Example 15. Results are presented in FIG. 30.

Human TNFα binding is comparable for temperature treated WT TNF1 and thehumanized TNF13 and 14; for temperature treated WT TNF2 and thehumanized TNF15-19; human TNFα binding is decreased for TNF21 and 22,and to a less extent for TNF23, while no effect is observed for TNF20compared to the temperature treated WT TNF3.

Trivalent TNFα Nanobodies

Example 33: Formatting of Trivalent TNFα Specific Nanobodies

TNF1, TNF2, TNF3 and ALB1 were formatted to trivalent nanobodies. Asspacer between 2 building blocks either a 9AA GlySer linker (Table 19SEQ ID No 68) or a 30 AA GlySer linker (Table 19 SEQ ID No 69) was used.This generated the constructs of Table 30. Table 29 lists the sequencesof trivalent TNFα nanobodies (SEQ ID NOs: 91 to 94).

Example 34: Expression of Trivalent TNFα Specific Nanobody

Expression was performed as described in Example 5. The His(6)-taggednanobodies are purified on Immobilized Metal Affinity Chromatography(IMAC). The Ni-NTA resin (Qiagen) is processed according to themanufacturer's instructions. The extracts are incubated with the resinand incubated for 30 min at RT on a rotator. The resin is washed withPBS and transferred to a column. The packed resin is washed with PBS (1in 10 diluted). Pre-elute with 15 mM Imidazole. The nanobodies areeluted from the column using 25 mM Citirc Acid pH=4. The eluatedfractions are analyzed by spotting on Hybond Membrane and visualizationwith Ponceau. Fractions containing protein are pooled and furtherpurified on Cation exchange followed by size exclusion. Purifiedproteins are collected, filter sterilized, concentration determined andstored in aliquots at −20° C.

Characterization of Trivalent TNFα/SA Specific Nanobodies

Example 35: Expression Level

Expression levels were calculated and represented in Table 31.

Example 36: SDS-PAGE Analysis

SDS-PAGE was performed as described in Example 8. FIG. 31 represents theSDS-PAGE gel.

Example 37: Western Blot Analysis

Western Blot analysis was performed as described in Example 9. FIG. 32represents the Western Blot analysis.

Example 38: Bio-Assay

The assay was performed as described in Example 13.

The results of the bivalent nanobodies are summarized in Table 32. Basedon their potency to neutralize cytotoxic activity, the molecules areequally potent and comparable to their potency as bivalent molecules.

Example 39: Binding to Human Serum Albumin

Binding was assayed as described above except that series of differentconcentrations were used. Each concentration was injected for 4 minutesat a flow rate of 45 μl/min to allow for binding to chip-bound antigen.Next, binding buffer without analyte was sent over the chip at the sameflow rate to allow for dissociation of bound nanobody. After 15 minutes,remaining bound analyte was removed by injection of the regenerationsolution (25 mM NaOH).

From the sensorgrams obtained for the different concentrations of eachanalyte K_(D)-values were calculated via steady state affinity whenequilibrium was reached.

Results are summarized in Table 33. A decrease in affinity was observedfor the formatted ALB1 binder compared to the wild type ALB1. Theaffinity however is still in the range of 7.2-14 nM.

Example 40: Temperature Stability

Samples were analysed as described in Example 15.

OD280 nm

OD at 280 nm was measured and the concentration was calculated. Resultsare summarized in Table 34. A decrease in protein content is observedfor TNF24, TNF27 and TNF28 starting at 60° C., while for TNF25 and TNF26starting from 70° C.

Western Blot

Samples were analyzed for the presence of aggregates as described inExample 15.

ELISA

The ELISA to detect binding to human TNFα was essentially performed asdescribed above. Results are presented in FIG. 33. Human TNFα binding isdecreased for TNF24 and TNF27, starting from 60° C. and for TNF25, TNF26and TNF28 starting at 70° C.

Humanised Monovalent Nanobodies (Second Round)

Example 41: Identification of Non-Human Amino Acid Positions in TNFα andSerum Albumin Specific Nanobodies

FIG. 34 (TNF1), FIG. 35 (TNF2), FIG. 36 (TNF3) and FIG. 37 (ALB1)represent multiple sequence alignments (Clustal W 1.7) with DP51, DP53,DP54 and DP29 sequences. The mutated molecules were expressed andpurified as described above, yielding the sequences of Table 35 SEQ IDNOs: 95 to 104 (against TNF-alpha and human serum albumin,respectively).

Characterisation of Humanised Nanobody

Example 42: Expression Level

Table 36 represents calculated expression levels. Expression wasachieved with yields in the range of 0.5-2.7 mg/ml.

Example 43: SDS-PAGE

SDS-Page was performed as described in Example 8. FIG. 38 represents theSDS-Page gel.

Example 44: Western Blot

Western Blot analysis was performed as described in Example 9. FIG. 39represents the Western Blot results.

Example 45: Bio-Assay

The assay was performed as described in Example 13.

The results of the humanised nanobodies are summarized in Table 37. Thewildtype nanobodies and first round of humanised nanobodies are includedas reference.

Example 46: Biacore

The analysis was performed as described in Example 12. FIG. 40 showsBiacore results.

Example 47: Temperature Stability

Samples were analysed as described in Example 15.

OD280 nm

OD at 280 nm was measured and the concentration was calculated. Resultsare summarized in Table 38.

No significant decrease in protein concentration is observed for thehumanised TNF1 nanobodies (TNF29-30). A decrease in proteinconcentration is observed for humanised TNF2 (TNF31-32) and TNF3 (TNF33)starting at 80° C.

Western Blot

Samples were analyzed for the presence of aggregates as described inExample 15.

ELISA

The ELISA to detect binding to human TNFα was essentially performed asdescribed in Example 15. Results are presented in FIG. 41.

Human TNFα binding is comparable for WT TNF1 and the humanised TNF29 andTNF30; comparable for WT TNF2 and the humanised TNF31 and TNF32; andalso for WT TNF3 and humanised TNF33.

Comparative Example

In this Comparative Example, nine Nanobodies of the invention werecompared with three Nanobodies from WO 04/041862, called “V_(HH)#1A” or“1A”, “V_(HH)3E” or “3E” and “V_(HH)#3G” or “3G” respectively (SEQ IDNOS:1, 4 and 5 in WO 04/041862). The assay used was the cell based assayusing KYM-cells referred to in WO 04/41862 (see for example Example 1,under 3)). The results are mentioned in Table 39 below. As can be seen,the Nanobodies of the invention have an EC50 value in this assay that is18-fold better than the EC50 value of 3E, the best performing Nanobodyaccording to WO 04/041862.

Example 48: Generation of Trivalent Bispecific Humanized Nanobodies™

Trivalent bispecific Nanobodies were formatted and cloned in the E. coliexpression vector pAX054 first and then rescued through PCR and clonedin the pPICZαA expression vector.

Description of Escherichia coli Expression Vector

pAX54 is a derivative of pUC19. It contains the LacZ promoter whichenables a controlled induction of expression using IPTG. The vector hasa resistance gene for Ampicillin or Carbenicillin. The multicloningsites harbours several restriction sites of which SfiI and BstEII arefrequently used for cloning of Nanobodies™. The signal peptide is thegen3 leader sequence which translocates the expressed Nanobody™ to theperiplasm.

Description of Pichia pastoris Expression Vector

pPICZαA contains a pUC-derived origin of replication allowingpropagation in E coli. It contains the promoter of the Pichia pastorisAOX1 (alcohol oxidase 1) gene. This 942 bp promoter region (i) allowsmethanol-inducible, high-level expression of the gene of interest, and(ii) targets plasmid integration to the AOX1 locus followingtransformation of Pichia with vector DNA that is linearized within the5′ AOX1 promoter region. Note that pPICZα vectors do not contain a yeastorigin of replication and that, consequently, transformants can only beisolated if recombination occurs between the plasmid and the Pichiagenome. The vector specifies resistance to the antibiotic Zeocin in bothE. coli and Pichia pastoris host cells. The vector incorporates thesecretion signal of the Saccharomyces cerevisiae α-mating factorallowing for efficient secretion of most proteins to the culture medium.The initiation ATG in the α-factor signal sequence corresponds to thenative initiation ATG of the AOX1 gene. The multicloning site harboursseveral restriction sites of which Xho1/EcoR1 or Xho1/Not1 are typicallyused for fusion of the Nanobody™ coding sequences to the secretionsignal. The multicloning site is followed by the AOX1 transcriptiontermination region. More details on this expression vector can be foundon the website of Invitrogen(http://www.invitrogen.com/content/sfs/manuals/ppiczalpha_man.pdf).

Formatting Trivalent Nanobodies

Three separate PCR reactions were set up to amplify the N-terminal, themiddle and the C-terminal Nanobody™ subunit using the oligo combinationsindicated in the WPA-0012. The N-terminal Nanobody™ was amplified usingM13_rev/Rev_9GlySer_L108; the middle Nanobody™ was amplified usingFor_GlySer/Short and Rev_15BspEI_L108; the C-terminal Nanobody™ wasamplified using For_BspEI/M13_for. A PCR reaction of 1 μl plasmid DNA(50-100 ng), 1.5 μl forward primer (10 μM→300 nM), 1.5 μl reverse primer(10 μM→300 nM), 1 μl dNTPs (10 mM→0.2 mM), 5 μl buffer (10×→1×), 0.75 μlenzyme (3.5 U/μl→2.6 U/μl) and 39.25 μl H₂O with a total volume of 50 μlwas prepared. Primer sequences are given in Table 40. A PCR program wasstarted with 2 minutes at 94° C. A cycle of 30 seconds at 94° C., 30seconds at 50° C. and 1 minute at 72° C. was repeated 30 times andfollowed by 10 minutes at 72° C. Amplification was checked by separating5 μl of the PCR reaction on a 2% agarose gel. The PCR product waspurified using the QIAquick PCR Purification Kit according to themanufacturer's instructions. One column was used and eluted with 50 μlEB buffer. The N-terminal VHH fragment was prepared by incubating 50 μlDNA and 2 μl BamHI (10 U/μl) in the appropriate buffer recommended bythe manufacturer at 37° C. for 2 hours. Subsequently, 2 μl SfiI (10U/μl) was added and the mixture was incubated at 55° C. for 2 hours. Themiddle VHH fragment was prepared by incubating 50 μl DNA and 2 μl BamHI(10 U/μl) and 2 μl BspEI (10 U/μl) in the appropriate buffer recommendedby the manufacturer at 37° C. for 2 hours. The C-terminal VHH fragmentwas prepared by incubating 50 μl DNA with 2 μl BspEI (10 U/μl) in theappropriate buffer recommended by the manufacturer at 37° C. for 2hours. Subsequently, 2 μl BstEII (10 U/μl) was added and the mixture wasincubated at 60° C. for 1 hours. The previous digestion reactions wereseparated on a 2% agarose gel. The VHH bands (350-450 bp) were cut outof the gel and the DNA was purified using the QIAquick Gel ExtractionKit according to the manufacturer's instructions. One column (with amaximum of 400 mg agarose gel per column) was used and the bound DNA waseluted with 50 μl EB buffer. DNA concentration was determined bymeasuring OD260 (1 OD unit=50 μg/ml). A ligation mixture with a finalvolume of 10 μl containing 100 ng vector pAX54, 12 ng N-terminal VHH, 12ng middle VHH fragment, 12 ng C-terminal VHH fragment, 1 μl ligationbuffer and 1 μl ligase (3 U) was prepared and incubated for 2 hours atroom temperature. Transformation of E. coli, TG1 was performed by using2 μl of ligation mixture. Colonies are analysed using PCT as describedin WPA-0010. Sequence analysis is performed on positive clones. Plasmidpreparation was performed using the Qiaprep spin Miniprep kit (Qiagen)according to the manufacturer's instructions and described above.Sequencing was performed at the VIB sequence facility, Antwerp, Belgium.

Amplification of Coding DNA

The Nanobody™ coding region cloned in the pAX054 E. coli expressionvector is rescued through PCR using an appropriate primer pair. Toensure that the Nanobody™ is expressed with a native N-terminus, thecoding region is cloned in frame with the Kex2 cleavage site of thesecretion signal. The forward primer fuses the C-terminal part of thesecretion signal, up to the Xho1 recognition site, to the Nanobody™coding region. A PCR reaction of 1 μl plasmid DNA (50-100 ng), 1.5 μlforward primer (10 μM→300 nM), 1.5 μl reverse primer (10 μM→300 nM), 1μl dNTPs (10 mM→0.2 mM), 5 μl buffer (10×→1×), 0.75 μl enzyme (3.5U/μl→2.6 U) and 39.25 μl H₂O with a total volume of 50 μl was prepared.Primer sequences are given in Table 41. A PCR program was started with 2minutes at 94° C. A cycle of 30 seconds at 94° C., 30 seconds at 50° C.and 2 minutes at 72° C. was repeated 20 times and followed by 10 minutesat 72° C. Amplification was checked by separating 5 μl of the PCRreaction on a 2% agarose gel. The PCR product was purified using theQIAquick PCR Purification Kit according to the manufacturer'sinstructions. One column was used and the bound DNA was eluted with 50μl EB buffer.

Cloning Strategy

The DNA fragment coding for the NB as well as the pPICZαA expressionvector is digested with the appropriate restriction enzymes (XhoI+NotI).The insert is obtained by incubating 50 μl PCR product with 2 μl XhoI(10 U/μl) and 2 μl NotI (10 U/μl) in the appropriate buffer recommendedby the manufacturer for 3 hrs at 37° C. Vector is obtained similarly,adapting the amount of restriction enzymes to the amount of plasmid.Both the vector and the NB coding fragment are purified and the DNAconcentration is quantified using the BioPhotometer (Eppendorf). Thefragment and the acceptor vector are ligated in equimolar ratio's using1 Unit T4 ligase (Promega) for 30 minutes at room temperature orovernight at 16° C. The DNA (20-30 ng) is transformed to TG1 cells.Colonies are analysed through PCR using the 3′AOX1 R and 5′AOX1 Fprimers. Sequence analysis is performed on positive clones. TNF30, TNF33and ALB8 were formatted to trivalent bispecific Nanobodies™. As spacerbetween the building blocks a 9AA GlySer linker was used.

Transformation P. pastoris

To isolate plasmid DNA, a preculture is started by inoculating a singlecolony of the clone in 50 ml Luria Broth+Ampicillin or Carbenicillin(100 μg/ml)+2% glucose and incubation at 37° C. overnight. Plasmid DNAis prepared using the Plasmid Midi kit (Qiagen) according to themanufacturer's instructions. The DNA is linearized by incubating 30 μgplasmid DNA with 6 μl BstX1 (10 U/μl) in the appropriate bufferaccording to the manufacturer's instructions for 3 hrs at 45° C.Digested DNA is purified using the PCR Purification kit (Qiagen)according to the manufacturer's instructions. The DNA is concentratedusing EtOH precipitation according to standard procedures. X-33electrocompetent cells are transformed with 10 μg linearized DNA andcells are allowed to grow for 48 hrs on a selective YPD agar platecontaining Zeocin (100/250/500 μg/ml). X-33 is a wild type Pichiapastoris strain; the strain itself as well as the derived recombinantstrains contain the native AOX1 gene and are able to metabolize methanol(Mut⁺).

Clones are screened for expression level by incubating single coloniesin 1 ml BGCM in a 24-well plate and growing them for 48 hrs at 30° C. at120 rpm. Cells are centrifuged and fresh BGCM is added to the cells forgrowth at 30° C. at 120 rpm during 48 hrs. Next, MeOH is added to afinal concentration of 0.5% and cells are grown at 30° C. at 120 rpmduring 8 hrs, after which MeOH is added again to a final concentrationof 0.5%. Cells are grown overnight at 30° C. at 120 rpm. Cells arecentrifuged and the supernatant is harvested and analysed in ELISA asdescribed in example 10.

Example 49: Expression and Purification of Trivalent BispecificHumanized Nanobodies™

Production in Pichia pastoris

Composition of buffers, solutions and others can be found on the websiteof Invitrogen(http://www.invitrogen.com/content/sfs/manuals/ppiczalpha_man.pdf).

A preculture was started by inoculating a single colony from plate in 5ml YPD. The culture was grown overnight at 180 rpm and 30° C. The nextday, the pre-culture was diluted to 50 ml of YPD and grown overnight at180 rpm and 30° C. Production cultures were started by inoculating thepre-culture to a final OD600 nm=0.04-0.08. Cultures were grown in BGCMfor 24 hrs at 30° C. at 180 rpm and centrifuged at 4,500 rpm for 30minutes. Cells were resuspended in 1/3 of the original volume in BGCMmedium with a final OD600 nm=15-20. Cells were induced with MeOH atregular time points, typically 3 times/day, never exceeding the 1% MeOHcontent. After 50 hours of induction the supernatant is harvested.Purification of Nanobody Expressed in Pichia pastoris

Culture supernatant is filtered over a 0.22 μm filtration membrane Microfiltration (Hydrosart, Sartorius). Sample is concentrated usingdiafiltration on 10 kDa ultra filtration membrane (HydroSart, Sartorius)and concentrated to 0.5-1 L.

Nanobodies™ are purified using Protein A affinity chromatography(MabSelect Xtra, GE Healthcare) using PBS as running buffer and Glycine[100 mM pH=2.5] for elution. Samples are neutralized using 1.5 M TrispH=8.8. Nanobodies™ are further processed in Anion ExchangeChromatography (Source 30Q, GE Healthcare). Samples are diluted 10-foldwith 10 mM Piperazine pH=10.2 and adjusted to pH=10.2 with 1M NaOH and aconductivity of <2 mS/cm with MilliQ water.Nanobodies are processed in Size Exclusion chromatography (Superdex 75pg, Hiload XK26/60, GE Healthcare) and LPS is removed via Anion ExchangeChromatography (Source 30Q, GE Healthcare) by passage through 5 mlcolumn, which is sanitized with 1M NaOH and equilibrated in DulbeccoPBS.

To determine the purity, protein samples were analyzed on a 15% SDS-PAGEgel as described in example 8. The gel is processed using theSilverQuest™ according to general procedures described by themanufacturer (Invitrogen). Alternatively, gel is processed usingcoomassie brilliant blue or in western blot as described in example 8and 9. Results are given in FIG. 42.

Example 50: Characterization of Trivalent Bispecific HumanizedNanobodies™

TNF60 consists of 363 amino acids. The protein has a molecular weight of38,441 Da. The pI is 8.71. The extinction coefficient at 280 nm is1.736.

Mass Spectrophotometry

The mass of the protein was determined in ESI-MS according to standardprocedures. The theoretical mass of TNF60 is 38,441 Da. The protein has2 S-S bridges which should result in a mass of 38,435 Da in ESI-MS. Themass that was experimentally determined for TNF60 derived from 3different batches ranges from 38,433 Da to 38,435 Da, differingmaximally 0.005% with the theoretical mass.

N-Terminal Sequencing

N-terminal sequencing was performed by Edman degradation according tostandard procedures. N-terminal sequencing showed that the proteinsequence for the first 7 amino acids is as follows: EVQLVES (SEQ ID NO:487). This is consistent with the theoretical protein sequence, whichindicates proper N-terminal processing.

Analytical Sizing

Samples (100 ug) were analysed on the high resolution Superdex75 column,to characterize the different batches of Nanobody™. Size exclusionchromatography of the Nanobody™ typically yields a symmetrical peak,with a retention time of 11.5 min on Superdex75. The absorbance istypically recorded at 280, 254 and 214 nm. The 214 nm measurementpermits higher detection sensitivity. Analytical sizing in PBS providesa symmetrical peak. No contaminants were observed. The retention timeobserved for 3 different batches is 11.5-11.55 min. A representativeprofile is shown in FIG. 43.

Example 51: Binding of TNF60 to Human TNFα in ELISA

The functionality of TNF60, i.e. binding to human TNFα was analyzed inELISA as described in example 10. The results are summarized in FIG. 44and clearly demonstrate a dose-dependent and saturable binding of 2batches of TNF60 to human TNFα.

Example 52: Functionality in Cell-Based Assay

The potency to neutralize the cytotoxic activity of TNFα was analyzed ina cell-based assay as described in example 13. The results aresummarized in Table 42 and in FIGS. 45 and 46.

The data show that TNF60 has potency in the range of Enbrel/Etanerceptand a 10-fold better potency than Humira/Adalimumab andRemicade/Infliximab.

Example 53: Binding of TNF60 to Serum Albumin

Binding to human and rhesus serum albumin was analyzed in Biacore asdescribed in example 12. KD, kon and koff values are represented inTable 43. TNF60 is compared to TNF24, which is the trivalent bispecificparent Nanobody™ with wild-type building blocks.

Affinity of TNF60 for human and rhesus serum albumin is similar.Affinity is 2-fold lower as compared to the affinity observed for TNF24which is the wild type analogue of TNF60. K_(on) is identical for bothmolecules, but the k_(off) is 2-fold higher for TNF60.

Example 54: Pharmacokinetic and Immunogenicity Analysis of TrivalentBispecific Humanized Nanobodies in Mice

Animals

DBA1 or BALBc mice were warmed up under an infrared lamp and 200 μlNanobody™ (100 μg per mouse) was injected intravenously in the tail.Blood samples were obtained at different time points by making a smallincision in the tail and collecting the blood in a microtube. Typically,blood was sampled at t=15 min, 2 hrs, 4 hrs, 6 hrs, 1 day, 2 days, 3days, 4 days, 7 days, 14 days. Serum was prepared according to standardprocedures.

Determination of Nanobody™ Concentration in Mouse Serum

A microtiterplate (NUNC, Maxisorb) was coated with 2 μg/ml neutravidinovernight at 4° C. The plate was washed 5 times with PBS/0.05% Tween-20and blocked for 2 hours at RT with PBS/1% casein. Biotinylated humanTNFα (1/2000 in PBS/0.2% casein; 400 ng/ml) was applied to the wells andincubated for 1 hr at RT. The standard reference Nanobody™ was appliedstarting at a concentration of 5 μg/ml and using 5-fold dilutions in PBScontaining 1% mouse plasma. The Nanobodies™ were allowed to bind for 2hours at RT. The plate was washed 5 times and rabbit polyclonalanti-Nanobody (R23) was applied at a 2000-fold dilution for one hour atRT. After washing of the plate, binding was detected withgoat-polyclonal-anti-rabbit-HRP (DAKO) at a 3000-fold dilution for onehour at RT, and stained with ABTS/H₂O₂. The OD405 nm was measured.

This first ELISA was used to determine the linear range of the standardreference. In a second ELISA, the standard reference was used atconcentrations in this linear range and typically using 2-folddilutions. In this second ELISA, serum test samples were diluted100-fold and further 5-fold dilutions were made in 1% mouse plasma, todetermine the dilution at which the serum samples provide a read-out inthe linear range of the standard curve. In a third ELISA, serum samplesare diluted at an appropriate concentration determined in the secondELISA and using 2-fold dilutions for accurate determination of theNanobody™ concentration in the serum samples.

Experiments were performed to determine the pharmacokinetic profile ofTNF60 in mice (n=3). A Cmax value of 103.84±31 μg/ml was reached 15minutes after administration. The half-life (t1/2β) was determined to be1.9 days, similar to the half-life of mouse serum albumin, indicatingthat TNF60 adopts the half-life of serum albumin. Data are presented inFIG. 47.

Determination of Anti-Nanobody Antibodies in Mice

Nanobody™ was coated at 5 μg/ml in PBS at 4° C. overnight. The plate waswashed 5 times with PBS/0.05% Tween-20 and blocked for 2 hours at RTwith PBS/1% casein. Serum samples were diluted 100-fold and applied tothe wells for incubation during 1 hr at room temperature. Detection wasperformed using 1000-fold diluted polyclonal rabbit anti-mouse-HRP(DAKO, P0260) and using ABTS as substrate.

Serum samples were diluted 50-fold and analyzed for the presence ofmouse anti-TNF60 antibodies. Lack of immunogenicity was demonstrated forTNF60. Data are presented in FIG. 48.

Example 55: Generation of Bivalent Long Half-Lived Humanized Nanobodies™

Description of Pichia pastoris Expression Vector

See example 48

Formatting Bivalent Nanobodies™

Two separate PCR reactions were set up to amplify the N-terminal and theC-terminal Nanobody™ subunit using the procedures as indicated in theWPA-0011. For the amplification of the N-terminal Nanobody™ PiForLongand Rev_30GlySer_L108 were used as primer combination; for theamplification of the C-terminal Nanobody™ For_GlySer and PiRevCys1humwere used or alternatively For_GlySer and PiRevCys2hum, introducing therestriction sites required for formatting and free cysteine residuesrequired for C-terminal modifications.

PCR reaction of 1 μl plasmid DNA (50-100 ng), 1.5 μl forward primer (10μM→300 nM), 1.5 μl reverse primer (10 μM→300 nM), 1 μl dNTPs (10 mM→0.2mM), 5 μl buffer (10×→1×), 0.75 μl enzyme (3.5 U/μl→2.6 U/μl) and 39.25μl H₂O with a total volume of 50 μl was prepared. Primer sequences aregiven in Table 44. A PCR program was started with 2 minutes at 94° C. Acycle of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72°C. was repeated 30 times and followed by 10 minutes at 72° C.Amplification was checked by separating 5 μl of the PCR reaction on a 2%agarose gel. The PCR product was purified using the QIAquick PCRPurification Kit according to the manufacturer's instructions. Onecolumn was used and eluted with 50 μl EB buffer. The N-terminal VHHfragment was prepared by incubating 50 μl DNA and 2 μl BamHI (10 U/μl)and 2 μl XhoI (10 U/μl) in the appropriate buffer recommended by themanufacturer at 37° C. for 1.5 hours. The C-terminal VHH fragment wasprepared by incubating 50 μl DNA and 2 μl BamHI (10 U/μl) and 2 μl EcoRI(10 U/μl) in the appropriate buffer recommended by the manufacturer at37° C. for 1 hour. The previous digestion reactions were separated on a2% agarose gel. The VHH bands (350-450 bp) were cut out of the gel andthe DNA was purified using the QIAquick Gel Extraction Kit according tothe manufacturer's instructions. One column (with a maximum of 400 mgagarose gel per column) was used and the bound DNA was eluted with 50 μlEB buffer. DNA concentration was determined by measuring OD260 (1 ODunit=50 μg/ml). A ligation mixture with a final volume of 10 μlcontaining 100 ng vector pPICZαA, linearized with XhoI/EcoRI, 30 ngN-terminal VHH, 30 ng C-terminal VHH fragment, 1 μl ligation buffer and1 μl ligase (3 U) was prepared and incubated for 1 hour at RT.Transformation of E. coli, TG1 was performed by using 2 μl of ligationmixture. Colonies are analysed using PCR as described in WPA-0010 butusing the AOXIFor/AOXIRev primer combination. Sequence analysis isperformed on positive clones. Plasmid preparation was performed usingthe Qiaprep spin Miniprep kit (Qiagen) according to the manufacturer'sinstructions and described above. Sequencing was performed at the VIBsequence facility, Antwerp, Belgium.

Transformation of P. pastoris

See example 48.

TNF30 was formatted to bivalent Nanobodies™. As spacer between the 2building blocks a 30 AA GlySer linker was used. To allow for C-terminalsite-specific modifications a free cysteine was introduced, either asthe last AA of the Nanobody™ or with an extra spacer consisting ofGlyGlyGlyCys (SEQ ID NO: 471).

Example 56: Expression and Purification of Bivalent Long Half-LivedHumanized Nanobodies™

Production in Pichia pastoris

See example 49

Purification of Bivalent Nanobodies

The culture medium was made cell-free via centrifugation and 0.22 μmfiltration. The sterile medium was stored at 4° C. until furtherprocessed. Low molecular weight contaminants were reduced via ultrafiltration on a 10 kDa ultra filtration (UF) membrane (HydroSartSartocon Slice Cassette, Sartorius) as follows: four liter medium wasconcentrated to 0.5-1 lit, then diluted with 5 lit PBS and againconcentrated to 0.5 lit. This action was carried out twice.

The retentate of the UF was filtered through nylon 47 mm membranes 0.45μm (Alltech #2024).

In a next step bivalent Nanobody™ was captured from the concentratedmedium via Protein A affinity purification (using MabSelectXtra™, GEHealthcare). The column [35×100 mm] was equilibrated in PBS and aftersample application washed extensively with PBS. TNF56 was eluted withGlycine [100 mM, pH=2.5].

The eluted fractions of MabSelectXtra™ were neutralized with Tris [1.5M,pH 8,8] and stored at 4° C. TNF56 was concentrated and purified via AEX(A=10 mM piperazin, pH 10.8 and B=1M NaCl in 50 mM Tris, pH 7.5) usingSource 30Q (GE Healthcare). To this end the Nanobody™ fractions werediluted with A buffer (10 mM piperazin, pH 10.8) to a conductivity of 5mS/cm and the pH was adjusted to 10.8. The column [25×100 mm] wasequilibrated in A buffer before loading the sample onto the column.TNF56 was eluted with a 5 Column Volume (CV) gradient. The pH of thecollected fractions was adjusted to 7.8 using 1M Tris pH=7.8.

Pegylation of Bivalent Nanobodies™ Expressed in Pichia pastoris

Reduction of C-Terminal Cysteines

Dithiotreitol (DTT, Aldrich Cat 15,046-0) was added to the neutralizedfractions to reduce potential disulfide bridges that formed between thecarboxy terminal cysteines of the Nanobodies™ (usually around 20%). Afinal concentration of 10 mM DTT and incubation overnight at 4° C. wasfound to be optimal. The reduction was evaluated by analytical sizeexclusion chromatography (SEC). Therefore 25 μl of the reduced Nanobody™was added to 75 μl D-PBS and injected on a Sup75 10/300 GL columnequilibrated in Dulbecco's PBS (D-PBS, Gibco™ REF 14190-094).

Non reduced Nanobody™ and DTT was removed by preparative SEC on a Hiload26/60 Superdex75 prep grade column equilibrated in D-PBS.

The concentration of the reduced Nanobody™ was measured by measuring theAbsorbance at 280 nm. A Uvikon 943 Double Beam UV/VIS Spectrophotometer(method: see SOP ABL-0038) was used. The absorption was measured in awavelength scan 245-330 nm. Two Precision cells made of Quartz Suprasil®cells were used (Hellma type No.: 104-QS; light path: 10 mm). First theabsorption of the blank was measured at 280 nm by placing two cellsfilled by 900 μl D-PBS. The sample was diluted (1/10) by adding 100 μlof the sample to the first cell. The absorption of the sample wasmeasured at 280 nm.

The concentration was calculated with following formula:

$\frac{{{OD}_{280}{Sample}} - {{OD}_{280}{blank}}}{ɛ \times 1} \times 10\mspace{14mu}\begin{matrix}{{{For}\mspace{14mu}{TNF}\; 55\text{:}\mspace{14mu} ɛ} = 1.85} \\{{{For}\mspace{14mu}{TNF}\; 56\text{:}\mspace{14mu} ɛ} = {1.83.}}\end{matrix}$PEGylation

To PEGylate Nanobody™ a 5× molar excess of freshly made 1 mM PEG40solution was added to the reduced Nanobody™ solution. (MPEG2-MAL-40K ofNEKTAR™

Transforming Therapeutics (2D3YOTO1) Mw=40,000 g/mol; MPEG2-MAL-60K ofNEKTAR™ Transforming Therapeutics (2D3YOVO1) Mw=60,000 g/mol).

The Nanobody™-PEG mixture was incubated for 1 h at room temperature (RT)with gentle agitation and then transferred to 4° C. The PEGylation wasevaluated via analytical SEC. Therefore 250 of the Nanobody™ was addedto 750 D-PBS and injected on a Sup75HR 10/300 column equilibrated inD-PBS. Pegylated Nanobody™ eluted in the range of the exclusion volumeof the column (>75 kDa).

The PEGylated and non PEGylated Nanobody™ were separated via cationexchange chromatography (CEX, using Source30S, GE Healthcare; Abuffer=25 mM citric acid pH=4 and B=1M NaCl in PBS). The sample wasdiluted to a conductivity of <5 mS/cm and the pH was adjusted to 4.0.The column [25×100 mm] was equilibrated and after sample applicationwashed extensively with A-buffer. Pegylated Nanobody™ was eluted with a3 CV gradient. The collected Nanobody™ was buffer exchanged to D-PBS bySEC on a Hiload 26/60 Superdex 75 prep grade column equilibrated inD-PBS.

Finally the Nanobody™ was made LPS-free via passage over an anionexchange column (Source30Q). The column (10×100 mm) was sanitizedovernight in NaOH [1M] and afterwards equilibrated in endotoxin freeD-PBS.

Biotinylation

To biotinylate Nanobody™ a 5× Molar excess of biotin (EZ-Link®Maleimide-PO2-Biotin, Pierce #21901) from a 10 mM stock solution wasadded to the reduced Nanobody™ (see 5.5.1). The biotin-Nanobody™ mixturewas incubated for 1 h at RT with gentle agitation and then stored at 4°C.

The purity of biotinylated Nanobody™ was controlled via analytical SEC.Therefore 25 μl of biotinylated Nanobody™ was added to 75 μl D-PBS andinjected on a Sup75HR 10/300 column equilibrated in D-PBS. From theobtained chromatogram could be concluded that the Nanobody™-biotin needsno further purification: no dimerization of Nanobody™ via an oxidationof free sulfidrils could be detected. A buffer change to D-PBS was doneby a passage over a desalting column Sephadex G25 fine (90 ml) column.

Finally the Nanobody™-biotin was made LPS-free by passage over an anionexchange column (Source30Q, GE Healthcare). The column (1×10 cm) wassanitized overnight in 1M NaOH and then equilibrated in D-PBS.

To determine the purity, protein samples were analyzed on a 15% SDS-PAGEgel as described in example 8 and 49. Results are presented in FIGS. 49and 50.

Example 57: Characterization of Bivalent Long Half-Lived HumanizedNanobodies™

Biochemical Characterization

TNF55 consists of 260 amino acids. The protein has a molecular weight of27,106 Da. The pI is 8.67. The extinction coefficient at 280 nm is1.850.

TNF56 consists of 264 amino acids. The protein has a molecular weight of27,365 Da. The pI is 8.67. The extinction coefficient at 280 nm is1.830.

Mass Spectrophotometry

The theoretical mass of TNF55 is 27,106 Da. The TNF55-Biotine proteinhas 2 S-S bridges and a biotine modification which should result in amass of 27,627 Da in ESI-MS. The mass that was experimentally determinedfor TNF55-biotine is 27,627 Da.

The theoretical mass of TNF56 is 27,365 Da. The TNF55-Biotine proteinhas 2 S-S bridges and a biotine modification which should result in amass of 27,886 Da in ESI-MS. The mass that was experimentally determinedfor TNF55-biotine is 27,886 Da.

N-Terminal Sequencing

N-terminal sequencing of TNF56-PEG40 showed that the protein sequencefor the first 7 amino acids is as follows: EVQLVES (SEQ ID NO: 487).This is consistent with the theoretical protein sequence, whichindicates proper N-terminal processing.

Analytical Sizing

Analytical sizing of TNF56-PEG40 in PBS provides a symmetrical peak. Nocontaminants were observed. The retention time observed is 8.5 ml onSuperdex HR 75 and 10.32 ml on Superdex HR 200. A representative profileis shown in FIGS. 51 and 52.

Example 58: Functionality in Cell-Based Assay

The potency to neutralize the cytotoxic activity of TNFα was analyzed ina cell-based assay. Potency was examined at different concentrations ofNanobody™ as well as of the commercially available Enbrel, Humira andRemicade on a molar base. The higher the EC50 observed the lower theactivity of the compound to neutralize TNFα.

The results are summarized in Table 45 and FIGS. 53 and 54.

The data show an increase in potency for the bivalent Nanobodies™ whencompared to the monovalent Nanobody™ TNF1. Potency of TNF55 derivativesis similar to TNF56 derivatives, which is in the range of Enbrel and10-fold better than Humira and Remicade.

Example 59: Pharmacokinetic and Immunogenicity Analysis of Bivalent LongHalf-Lived Humanized Nanobodies in Mice

See example 54

Experiments were performed in order to examine the half-life ofpegylated Nanobodies™ in mice. The half-life of bivalent TNF56-PEG40 wascompared to the half-life of TNF56-PEG60. Both Nanobodies™ havecomparable half-life of ˜2 days. The results are presented in FIG. 55.

In addition, the half-life of pegylated bivalent 3E-3E was explored. Thehalf-life of 3E-3E-PEG20 was compared to the half-life of 3E-3E-PEG40after intravenous administration of 100 μg of the Nanobodies™.3E-3E-PEG20 has a half-life of 17 hrs, while 3E-3E-PEG40 has a half-lifeof 2.1 days, comparable to the half-life of 3E-3E-MSA21. The results arepresented in FIG. 56.

Serum samples were diluted 100-fold and analyzed for the presence ofmouse anti-TNF56-PEG40 or anti-TNF56-PEG60 antibodies. Lack ofimmunogenicity was demonstrated for both molecules. Data are presentedin FIG. 57.

Example 60: Efficacy of Anti-TNF-α Nanobody TNF60 (TNF60) in Preventionof Chronic Polyarthritis

Transgenic mouse lines carrying and expressing a 3′-modified humantumour necrosis factor (hTNF-alpha, cachectin) transgene were used as amodel to study the efficacy of TNF60 (TNF60) in preventing thedevelopment of arthritis (EMBO J. 10, 4025-4031). These mice have beenshown to develop chronic polyarthritis with 100% incidence at four toseven weeks of age.

From the third week of age, litters of transgenic mice were divided intogroups of eight animals. Before initializing the study, the average bodyweight was calculated for each group. From then on, during the wholestudy animal weights were recorded once a week for each group.

To test the efficacy of TNF60 in the prevention of chronicpolyarthritis, intraperitoneal injections were given twice a week toeach animal of a particular group according to the following scheme:

Group 1 (negative control): phosphate buffered saline (PBS) (formulationbuffer)

Group 2 (Nanobody treatment): TNF60 at a final dose of 30 mg/kg

Group 3 (Nanobody treatment): TNF60 at a final dose of 10 mg/kg

Group 4 (Nanobody treatment): TNF60 at a final dose of 3 mg/kg

Group 5 (1^(st) positive control): Enbrel at a final dose of 30 mg/kg

Group 6 (1^(st) positive control): Enbrel at a final dose of 10 mg/kg

Group 7 (2^(nd) positive control): Remicade at a final dose of 30 mg/kg

Group 8 (2^(nd) positive control): Remicade at a final dose of 10 mg/kg

Group 9 (2^(nd) positive control): Remicade at a final dose of 3 mg/kg

For each group, dates of injection and injection volumes were noted.

Injections continued for seven weeks. During this period, clinicalscores were recorded by observing macroscopic changes in jointmorphology for each animal.

At 10 weeks of age, all mice were sacrificed and sera and joints werecollected. Sera were stored at −70° C. and ankle joints were conservedin formalin.

For selected groups, ankle joints were embedded in paraffin andsectioned. Ankle joint sections were subsequently used forhistopathological evaluation of disease progression.

Results are depicted in FIG. 58.

Example 61: Efficacy of Anti-TNF-α Nanobody TNF60 (TNF60) in TherapeuticTreatment of Chronic Polyarthritis

Transgenic mouse lines carrying and expressing a 3′-modified humantumour necrosis factor (hTNF-alpha, cachectin) transgene were used as amodel to study the efficacy of TNF60 (TNF60) in therapeutic treatment ofarthritis (EMBO J. 10, 4025-4031). These mice have been shown to developchronic polyarthritis with 100% incidence at four to seven weeks of age.

From the sixth week of age, litters of transgenic mice were divided intogroups of eight animals. Before initializing the study, the average bodyweight was calculated for each group. From then on, during the wholestudy animal weights were recorded once a week for each group.

To test the efficacy of TNF60 in the therapeutic treatment of chronicpolyarthritis, intraperitoneal injections were given twice a week toeach animal of a particular group according to the following scheme:

Group 1 (negative control): phosphate buffered saline (PBS) (formulationbuffer)

Group 2 (Nanobody treatment): TNF60 at a final dose of 30 mg/kg

Group 3 (Nanobody treatment): TNF60 at a final dose of 10 mg/kg

Group 4 (1^(st) positive control): Enbrel at a final dose of 30 mg/kg

Group 5 (2^(nd) positive control): Remicade at a final dose of 30 mg/kg

For each group, dates of injection and injection volumes were noted.

Injections continued for seven weeks. During this period, clinicalscores were recorded by observing macroscopic changes in jointmorphology for each animal.

At 13 weeks of age, all mice were sacrificed and sera and joints werecollected. Sera were stored at −70° C. and ankle joints were conservedin formalin.

For selected groups, ankle joints were embedded in paraffin andsectioned. Ankle joint sections were subsequently used forhistopathological evaluation of disease progression.

Results are depicted in FIG. 59.

Example 62: Effect of Formatting on Efficacy of an Anti-TNF-α Nanobodyin Prevention of Chronic Polyarthritis

Transgenic mouse lines carrying and expressing a 3′-modified humantumour necrosis factor (hTNF-alpha, cachectin) transgene were used as amodel to study the efficacy of an anti-TNF-α Nanobody formatted indifferent ways in the prevention of chronic polyarthritis (EMBO J. 10,4025-4031). These mice have been shown to develop chronic polyarthritiswith 100% incidence at four to seven weeks of age.

From the third week of age, litters of transgenic mice were divided intogroups of eight animals. Before initializing the study, the average bodyweight was calculated for each group. From then on, during the wholestudy animal weights were recorded once a week for each group.

To study the efficacy of an anti-TNF-α Nanobody in different formats forprevention of chronic polyarthritis, intraperitoneal injections weregiven twice a week to each animal of a particular group according to thefollowing scheme:

Group 1 (negative control): phosphate buffered saline (PBS) (formulationbuffer)

Group 2 (Nanobody format 1): TNF60 at a final dose of 10 mg/kg

Group 3 (Nanobody format 1): TNF60 at a final dose of 2.5 mg/kg

Group 4 (Nanobody format 1): TNF60 at a final dose of 1 mg/kg

Group 5 (Nanobody format 2): TNF56-PEG40 at a final dose of 10 mg/kg

Group 6 (Nanobody format 2): TNF56-PEG40 at a final dose of 1.8 mg/kg

Group 7 (Nanobody format 2): TNF56-PEG40 at a final dose of 0.7 mg/kg

Group 8 (Nanobody format 3): TNF56-biot at a final dose of 1.8 mg/kg

Group 9 (Nanobody format 4): TNF30 at a final dose of 1 mg/kg

Group 10 (Nanobody format 5): TNF1 at a final dose of 1 mg/kg

Group 11 (1^(st) positive control): Enbrel at a final dose of 10 mg/kg

Group 12 (2^(nd) positive control): Remicade at a final dose of 10 mg/kg

For each group, dates of injection and injection volumes were noted.

Injections continued for seven weeks. During this period, clinicalscores were recorded by observing macroscopic changes in jointmorphology for each animal.

At 10 weeks of age, all mice were sacrificed and sera and joints werecollected. Sera were stored at −70° C. and ankle joints were conservedin formalin.

For selected groups, ankle joints were embedded in paraffin andsectioned. Ankle joint sections were subsequently used forhistopathological evaluation of disease progression.

Results are depicted in FIG. 60.

Example 63: Pharmacokinetic Study of Anti-TNF-α Nanobodies TNF60 (TNF60)and TNF56-PEG40 in Rhesus Monkey

Captive-bred rhesus monkeys (Macaca mulatta) are used to determine thepharmacokinetic profile of TNF60 and TNF56-PEG40.

Sixteen animals are used in this study (eight males and eight females)and are divided into four groups (two males and two females per group).All animals weighed approximately 5 kg and are disease-free for at leastsix weeks prior to use. Sniff® Pri vegetarisch V3994 serves as food.Sixty g/kg b.w. are offered to each monkey. The residue is removed. Atregular intervals (at least twice a year) the food is analyzed based onEPA/USA for contaminants by LUFA-ITL. Tap water is offered ad libitum.The animals in each treatment group are housed in a block of severaladjacent cages within the monkey unit. The monkeys are kept singly inV₂A steel cages with a size of 90 cm×82 cm×96 cm. The room temperatureis maintained at 23° C.±3° C. (maximum range) and the relative humidityat 60%±20% (maximum range). Deviations from the maximum range caused forexample during cleaning procedure are dealt with in SOPs. The rooms arelit and darkened for periods of 12 hours each.

Two groups are infused with TNF60 and two groups are infused withTNF56-PEG40. Intravenous infusions of TNF60 and TNF56-PEG40 (dissolvedin PBS) into the vena cephalica of the right or the left arm usingindwelling catheters and a TSE infusion pump (see below) are given at afixed dose of 2 mg/kg.

Four single administrations are performed, separated by a wash-outperiod of at least fourteen days. After the last administration thefollow-up period is at least eight weeks. Two out of the four groups aretreated with TNF60 or TNF56-PEG40 in combination with methotrexate (MTX)(dissolved in PBS). Group 2 is treated with TNF60 and MTX; group 4 istreated with TNF56-PEG40 and MTX. MTX is dosed weekly intramuscularly at0.2 mg/kg. On the administration days, MTX is given approximately 30minutes prior to administration. Dosing starts at the first Nanobodyadministration and will continue throughout the eight week wash-outafter the fourth dose. There are fourteen single MTX administrations,separated by a wash-out period of at least one week starting at thefirst test item administration.

Example 64: Synovium-Derived Fibroblast Studies

In this study the ability of the anti-TNF biologicals, ALX0071 andEtanercept, to attenuate TNFα-induced IL-6 production by RA-synoviumderived fibroblasts was assessed.

Isolation of Synovial Fibroblasts

Synovial joint tissue from consenting RA patients was stored inDMEM-based medium with antibiotics at 4° C. for up to 96 hours afterjoint replacement surgery. Synovial cells were isolated from dissectedsynovium by collagenase digestion at 37° C. for 2 hours. The resultantcell suspension was then washed by a series of centrifugation andresuspension steps and the resultant cells then cultured at 37° C. inDMEM-based culture medium supplemented with 10% FCS (v/v). The resultantfibroblasts were used for the following experiment at either the secondor third passage. Cells from four donors were used in individualexperiments. Fibroblasts were seeded into 96-well flat bottompolystyrene plates at 1.5×10⁴ cells in a final volume of 250 μL ofDMEM-based culture medium supplemented with 10% FCS (v/v) per well andcultured overnight.

Stimulation of Synovial Fibroblasts

Cells were then incubated for 72 hours in DMEM-based culture mediumsupplemented with TNFα at 50 ng per mL (3 nM (R&D Systems 210-TA/CF)alone or in the presence of increasing doses of ALX0071 (0.575 to 1920ng per mL; 0.015 to 50 nM) or Etanercept (Wyeth Labs; 3.75 to 11250 ngper mL; 0.025 to 75 nM). The final volume in each well was 250 μL andeach assessment was performed in triplicate. After 72 hours, thesupernatant media was removed and stored at −40° C. prior to analysis byIL-6 ELISA (R&D Systems). The inhibition of TNFα-induced IL-6 productionwas determined and IC₅₀ values were calculated for both ALX0071 andEtanercept.

Summary of Results

Both ALX10071 and Etanercept dose-dependently reduced TNF□-induced IL-6production by RA synovium derived fibroblasts from all four donors.There was a similar potency between the two reagents under these assayconditions.

Example 65: Murine Air Pouch Studies

In this study the ability of the anti-TNF biologicals, ALX0071 andEtanercept, to attenuate TNFα-induced cell infiltration in to a murineair pouch was assessed.

Creation of Air Pouch

Air pouches were formed by the sub-cutaneous (s.c.) injection of 2.5 mLof sterile air in to the dorsal surface of anaesthetised male C57B1/6/Jmice (25-30 g, Harlan). The pouch was re-inflated by injecting 2.5 ml ofsterile air 3 days later.

TNFα Stimulation

Six days after the initial creation of the air pouch, the animals wereanaesthetised and the pouch injected with 1 ml of 0.5%CarboxyMethylCellulose (CMC) vehicle containing 0.1 μg recombinant humanTNFα (R & D Systems, 210-TA-050/CF). In three other groups of animals,ALX0071 (0.0625, 0.125 and 0.25 mg/kg) was injected s.c. 19 hours priorto the injection of TNFα. A second three groups of animals were injected(s.c.) with Etanercept (Wyeth Labs, 0.125, 0.25 and 0.5 mg/kg)immediately prior to the injection of TNFα.

24 hours following TNFα □ injection, mice were culled with a risingconcentration of CO₂. Pouches were lavaged with 2 ml of ice coldendotoxin free sterile PBS containing 5 IU/ml heparin. Volumes wererecorded and 0.5 ml aliquots were separated for counting of the totalwhite blood cell (WBC) population on a Sysmex XT-Vet cell counter. Themean and standard error of the mean (SEM) total WBC counts for eachgroup were calculated per ml of lavage fluid withdrawn. Statisticalanalysis was by ANOVA with Kruskal-Wallis post-test on untransformeddata.

Summary of Results

Both ALX0071 and Etanercept attenuated the TNFα-induced WBC infiltrationin to the air pouches (Table). While this attenuation reachedstatistical significance at both the 0.125 (P<0.01) and 0.25 mg/kg(P<0.05) ALX0071 dose groups, statistical significance was not observedwith any Etanercept dose group.

TABLE 8 Name SEQ ID NO Sequence PMP1C2(TNF1) 52QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA LYYCARSPSGFNRGQGTQVTVSSPMP1G11 53 QVQLQESGGGMVQPGGSLRLSCAASGFDFGVSWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKTTLYLQMNSLKPEDTA LYYCARSPSGSFRGQGTQVTVSSPMP1H6 54 EVQLVESGGGLVQPGGSLRLSCATSGFDFSVSWMYWVRQAPGKGLEWVSEINTNGLITKYVDSVKGRFTISRDNAKNTLYLQMDSLIPEDTA LYYCARSPSGSFRGQGTQVTVSSPMP1G5(TNF2) 55 QVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAPGKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS PMP1H2 56QVKLEESGGGLVQPGDSLRLSCAASGRTFSDYSGYTYTVGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFTISRDIAKNTVDLLMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS PMP3G2 57AVQLVESGGGLVQPGDSLRLSCAASGRTFSDYSGYTYTVGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFTISRDIAKNTVDLLMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS PMP1D2 58AVQLVDSGGGLVQAGGSLRLSCAASGRTFSAHSVYTMGWFRQAPGKEREFVARIYWSSANTYYADSVKGRFTISRDNAKNTVDLLMNCLKPEDTAVYYCAARDGIPTSRSVEAYNYWGQGTQVTVSS PMP3D10 59QVQLVESGGGLVQAGGSLSLSCAASGRSFTGYYMGWFRQAPGKERQLLASISWRGDNTYYKESVKGRFTISRDDAKNTIYLQMNSLKPEDTAVYYCAASILPLSDDPGWNTNWGQGTQVTVSS PMP5F10(TNF3) 60EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERELLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSS PMP6A8(ALB2) 61AVQLVESGGGLVQGGGSLRLACAASERIFDLNLMGWYRQGPGNERELVATCITVG.DSTNYADSVKGRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIRRTWHSELWGQGTQVTVSS PMP6B4 62EVQLVESGGGLVQEGGSLRLACAASERIWDINLLGWYRQGPGNERELVATITVG.DSTSYADSVKGRFTISRDYDKNTLYLQMNSLRPEDTG LYYCKIRRTWHSELWGQGTQVTVSSPMP6A6(ALB1) 63 AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSSPMP6C1 64 AVQLVDSGGGLVQPGGSLRLSCAASGFSFGSFGMSWVRQYPGKEPEWVSSINGRGDDTRYADSVKGRFSISRDNAKNTLYLQMNSLKPEDTA EYYCTIGRSVSRSRTQGTQVTVSSPMP6G8 65 AVQLVESGGGLVQPGGSLRLTCTASGFTFRSFGMSWVRQAPGKDQEWVSAISADSSTKNYADSVKGRFTISRDNAKKMLYLEMNSLKPEDTA VYYCVIGRGSPSSPGTQVTVSSPMP6A5 66 QVQLAESGGGLVQPGGSLRLTCTASGFTFGSFGMSWVRQAPGEGLEWVSAISADSSDKRYADSVKGRFTISRDNAKKMLYLEMNSLKSEDTA VYYCVIGRGSPASQGTQVTVSSPMP6G7 67 QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMYWVRVAPGKGLERISRDISTGGGYSYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCAKDREAQVDTLDFDYRGQGTQVTVSS NC55TNF_S1C4 105EVQLVESGGGLVQAGDSLRLSCAASQIIFGSHVAAWFRQAPGREREFVAEIRPSGDFGPEGEFEHVTASLKGRFTIAKNSVDNTVYLQMNSLKPEDTAVYYCAAAPYRGGRDYRWEYEYEYWGQGTQVTV NC55TNF_S1C3 106EVQLVESGGGLVQPGGSLRLSCKNAGSTSNAYATGWFRRAPGKEREFVAGIQWSGGDAFYRNSVKGRFRITRDPDNTVYLQMNDLKPEDTAIYYCAQKLSPYYNDFDSSNYEYWGQGTQVTV NC55TNF_S2C1 107EVQLVESGGDLVQPGGSLRLSCAVSGQLFSTNDVGWYRRAPGKQRELVATITDDGTTDYGDDVKGRFVISREGEMVYLEMNSLKPEDTAVYYCNINRLRSTWGIRYDVWGQGTQVTVSS NC55TNF_S2C5 108EVQLVESGGGLVQPGGSLRLSCVVSGFTFSTTSMTWVRQAPGKFEEWVSFINSDGSSTTYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTA MYYCGRRGYGRDRSKGIQVTVASNC55TNF_S3C7 109 EVQLVESGGGTVQAGDSLRLSCAASGRSFSSVAMGWFRQAPGKQREFLAGVGYDGSSIRYAESVKGRFTIARGNRESTVFLQMENLKPEDTAVYFCTAEPIGAYEGLWTYWGQGTQVTVSS NC55TNF_S3C1 110XXXXVESGGGLMQPGGSLKLSCAASGFMFSDSAMGWFRQAPGKEREFVATISWNGGSSSYADFVKGRFTISRDNAKNTVYLQMNGLTPQDTAIYYCAGSYSNGNPHRFSQYQYWGQGTQVTVSS NC55TNF_BMP1B2 111EVQLVESGGGLVQAGGSLRLSCAASGRTFGTYAMGWFRQAPGKEREFVAAISWGGGSIVYAESAKGRFTISRDNAKXTMYLQMDSLKPEDTAVYYCAAANNIATLRQGSWGQGTQVTVSS NC55TNF_BMP1D2 112EVQLVESGGELVQAGGSLKLSCTASGRNFVTYAMSWFRRAPGKEREFVASISWSGDTTYYSNSVKGRFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVVQVIDPSWSGVNLDDYDYLGSGTQVTVSS NC55TNF_BMP1E2 113EVQLVESGGRLVQPGGSLRLSCKNAGSTSNAYATGWFRRAPGKEREFVAGIQWSGGDAFYRNSVKGRFRITRDPDNTVYLQMNDLKPEDTAIYYCAQKLSPYYNDFDSSNYEYWGQGTQVTVSS NC55TNF_BMP1G2 114EVQLVESGGGLVQPGGSLRLSCAASATISSIVMLGWYRQAPGKQREWVASITIGSRTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YFCNAVPPRDDYWGQGTQVTVSSNC55TNF_BMP2A2 115 EVQLVESGGGLVQAGGSLRLSCAASGQTSSSYDMGWFRQAPGEGREFVARISGSDGSTYYSDRAKDRFTISRDNTKNMVYLQMDRLKPDDTAVYYCRVPRYENQWSSYDYWGQGTQVTVSS NC55TNF_BMP2C2 116EVQLVESGGGLVQPGGSLRLSCAASGSTFSTYDMSWVRQAPGKGLEWVSGIDSGGGSPMYVDSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCAKFSTGADGGSWYWSYGMDSWGKGTQVTVSS NC55TNF_BMP2F2 117EVQLVESGGGLVQAGDSLRLSCEASERSSNRYNMAWFRQAPGKEREFLARVDVSGGNTLYGDSVKDRFTVSRINGKNAMYLQMNNLKPEDTAIYYCAAGGWGTTQYDYDYWGQGTQVTVSS NC55TNF_NC10 118EVQLVESGGGLVQPGGSLRLSCVCVSSGCTFSAYSMTWVRQAPGKAEEWVSFINSDGSSTTYADSVNGRFKISRDNAKKTLYLQMNSLGPED TAMYYCQRRGYALDRGQGTQVTVSSNC55TNF_NC11 119 EVQLVESGGGLVQAGDSLTLSCASSGRGFYKNAMGWFRQPPGKEREFVASIKWNGNNTYYADSVRGRFTISRGNAKNTENTVSLQMNSLKPEDTADYYCAADSSHYSYVYSKAYEYDYWGQGTQVTVSS NC55TNF_NC1 120EVQLVESGGGLVQPGGSLRLSCVFSGFAFSASSMAWVRQAPGKYEEWVSFINSDGSSTTYADSVQGRFTISRDNAKNTLYLQMNSLKSEDTA MYYCGRRGYGRDRSQGIQVTVSSNC55TNF_NC2 121 EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGTITNYADSVKGRFTISRDNGKNTVHLQMNSLKPEDTAVYHCAVVQPYSGGDYYTGVEEYDYWGXGTQVTVSS NC55TNF_NC3 122EVQLVESGGGLVQPGGSLRLSCVVSGFTFSATSMTWVRQAPGKAEEWVSFINSDGSSTTYADSVKGRFTISRDNAKNTLYLQMDDLQSEDTA MYYCGRRGYGRDRSRGIQVTVSSNC55TNF_NC5 123 EVQLVESGGGLVQAGGSLRLSCAASGGAFSNYDVGWFRQAPGEGREIVARISGSGDSTYSSNRAKGRFTISRDNAKNTVYLQMNSLKREDTAVYYCRAARYNGTWSSNDYWGQGTQVTVSS NC55TNF_NC6 124EVQLVESGGGLVQPGGSLRLSCECVSSGCTFSAYSMTWVRQAPGKAEEFVSFINSDGSSTTYANSVNGRFKISRDNAKKTLYLQMNSLGPED TAMYYCQRRGYALDRGQGTQVTVSSNC55TNF_NC7 125 QVQLVESGGGLVQAGGSLRLSCTASGQTSSTADMGWFRQPPGKGREFVARISGIDGTTYYDEPVKGRFTISRDKAQNTVYLQMDSLKPEDTAVYYCRSPRYADQWSAYDYWGQGTQVTVSS NC55TNF_NC8 126EVQLVESGGGLVQPGGSLRLSCVVSGFTFSTTSMTWVRQAPGKFEEWVSFINSDGSSTTYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTA MYYCGRRGYGRDRSKGIQVTVSSNC55TNF_S2C2 127 EVQLVESGGGLVQPGGSLRLSCVASASGVKVNDMGWYRQAPGKERELVATITDDGRTNYEDFAKGRFTISRDNAKNTVYLQMNSLLPEDTAVYYCNARTYWAHLPTYWGQGTQVTVSS NC55TNF_S1C6 128EVQLVESGGGLVQAGGSLRLSCAASGRSFGSVAMGWFRQAPGKEREFVAAIGYDGNSIRYGDSVKGRFTISRDNIKNTMYLEMENLNADDTARYLCAAEPLARYEGLWTYWGQGTQVTVSS NC55TNF_S3C2 129EVQLVESGGGLVQAGASLRLSCTTSTRTNDRFNMAWFHQAPGKDREFVSRIDVAGYNTAYGDFVKGRFTVSRDSAENTVVLQMNSLRPEDTGVYYCAAGGWGISQSDYDLWGQGTQVTVSS

TABLE 9 nanobody Class Estimated koff (1/s) PMP5 F10 III 2.63E−04 PMP1G5 II 3.59E−04 PMP1 C2 I 4.39E−04 PMP1 G11 I 1.15E−03 PMP1 H6 I 2.14E−03PMP1 H2 II 3.65E−03 PMP3 G2 II 1.09E−02

TABLE 10 #AA differences/ Nanobody Germline sequence total #AA % AAidentity ALB1 DP51/DP53 13/87 85.1 ALB2 DP54 26/87 70.2 TNF1 DP51/DP53 6/87 93.2 TNF2 DP54 16/87 81.7 TNF3 DP29 18/87 79.4

TABLE 11 Nanobody Induction time Yield (mg/L) ALB1 short/37° C. 18 ALB2short/37° C. 4 TNF1 short/37° C. 8.3 TNF2 short/37° C. 5 TNF3 short/37°C. 0.8

TABLE 12 50% binding (ng/ml) ID Human TNFα Rhesus TNFα TNF1 12 12 TNF220 >3000 TNF3 18 16

TABLE 13 50% inhibition (ng/ml) ID Human TNFα Rhesus TNFα TNF1 530 220TNF2 3500 >5000 TNF3 100 100

TABLE 14 Human TNFα Kd (1/s) TNF1 1.05E−03 TNF2 1.33E−03 TNF3 3.02E−04

TABLE 15 Human Rhesus Mouse albumin albumin albumin ALB1 KD (nM) 0.57 0.52   6.5 ka (1/Ms) 1.11E+06 1.05E+06 1.11E+06 kd (1/s) 6.30E−045.46E−04 7.25E−03 ALB2 KD (nM) 0.092 0.036 15.7 ka (1/Ms) 8.15E+051.94E+06 1.95E+05 kd (1/s) 7.52E−05 7.12E−05 3.07E−03

TABLE 16 assay: L929s + Act D (5000 c/w) TNF: human TNFα @ 0.5 ng/mlEC₅₀ in nM relative potency Nanobody mean stdev # mean stdev TNF1 1C20.707 0.265 14 0.015 0.007 TNF2 1G5 1.412 0.622 14 0.007 0.002 TNF3 5F100.224 0.133 14 0.048 0.019 Enbrel 0.009 0.005 45 1.002 0.011 Humira0.079 0.043 39 0.097 0.069 Remicade 0.083 0.037 45 0.103 0.058 assay:L929s + Act D (5000 c/w) TNF: rhesus TNFα @ 0.5 ng/ml EC₅₀ in nMrelative potency Nanobody mean stdev # mean stdev TNF1 1C2 0.693 0.305 90.015 0.009 TNF2 1G5 >50 9 TNF3 5F10 0.602 0.283 9 0.017 0.010 Enbrel0.009 0.003 7 1 0.000 Humira 0.071 0.025 8 0.103 0.059 Remicade >6.7 7

TABLE 17 % Untreated RT 37° C. 50° C. 60° C. 70° C. 80° C. 90° C. TNF1100 98 98 98 98 95 92 90 TNF2 100 99 100 99 97 96 63 50 TNF3 100 96 9798 96 94 75 70 ALB1 100 101 102 101 100 64 94 90 ALB2 100 100 102 100100 28 8 17

TABLE 18 EC₅₀ in relative nanobody temp in ° C. nM # potency TNF1control 0.916 1 0.013 92#2302nr1.TNF1 RT 0.873 1 0.014 37 0.901 1 0.01350 0.908 1 0.013 60 0.891 1 0.013 70 1.218 1 0.010 80 2.655 1 0.004 905.797 1 0.002 TNF2 control 2.500 1 0.005 92#2302nr2.TNF2 RT 2.165 10.005 37 2.212 1 0.005 50 2.241 1 0.005 60 1.782 1 0.007 70 2.487 10.005 80 2.818 1 0.004 90 6.135 1 0.002 TNF3 control 0.278 1 0.04392#2302nr3.TNF3 RT 0.289 1 0.041 37 0.295 1 0.040 50 0.290 1 0.041 600.281 1 0.042 70 0.293 1 0.040 80 0.576 1 0.021 90 0.861 1 0.014

TABLE 19 Name SEQ ID NO Sequence GS9 68 GGGGSGGGS GS30 69GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS TNF1-GS9- 70QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE TNF1(TNF4)WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRGQGTQVTVSSGGGGSGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRG QGTQVTVSS TNF2-GS9- 71QVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP TNF2(TNF5)GKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSSGGGGSGGGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAPGKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF3-GS9- 72EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERE TNF3(TNF6)LLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERELLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAA SILPLSDDPGWNTYWGQGTQVTVSSTNF1-GS30- 73 QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE TNF1(TNF7)WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRGQGTQVTVSS TNF2-GS30- 74QVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP TNF2(TNF8)GKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAPGKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLKPEDTAVYYCAARDGIPTSRSVGSYN YWGQGTQVTVSS TNF3-GS30-75 EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERE TNF3(TNF9)LLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERELLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSS TNF30-30GS- 419EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE TNF30-C (TNF55)WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSC TNF30-30GS- 420EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE TNF30-gggCWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTA (TNF56)VYYCARSPSGFNRGQGTLVTVSSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSSgggC

TABLE 20 ID Format Linker TNF4 TNF1-TNF1 9 AA GlySer TNF5 TNF2-TNF2 9 AAGlySer TNF6 TNF3-TNF3 9 AA GlySer TNF7 TNF1-TNF1 30 AA GlySer TNF8TNF2-TNF2 30 AA GlySer TNF9 TNF3-TNF3 30 AA GlySer

TABLE 21 Nanobody ™ Induction time Yield (mg/L) TNF4 ON/28° C. 3.2 TNF5short/37° C. 5.5 TNF6 short/37° C. 1.19 TNF7 ON/28° C. 2.7 TNF8short/37° C. 6.6 TNF9 ON/28° C. 1.3

TABLE 22 50% inhibition (ng/ml) ID Human TNFα TNF4 13 TNF5 6.3 TNF6 30TNF7 16 TNF8 23 TNF9 18

TABLE 23 EC₅₀ in nM relative potency Nanobody mean stdev # mean stdevassay: L929s + Act D (5000 c/w) TNF: human TNFa @ 0.5 ng/ml TNF4 0.2360.049 4 0.033 0.012 TNF5 0.020 0.010 9 0.566 0.275 TNF6 0.078 0.047 80.179 0.168 TNF7 0.013 0.005 8 0.673 0.211 TNF8 0.007 0.002 2 1.2400.137 TNF9 0.012 0.005 6 0.729 0.242 Enbrel 0.009 0.005 45 1.002 0.011Humira 0.079 0.043 39 0.097 0.069 Remicade 0.083 0.037 45 0.103 0.058assay: L929s + Act D (5000 c/w) TNF: rhesus TNFa @ 0.5 ng/ml TNF4 0.1410.025 4 0.065 0.015 TNF5 35.000 16.000 5 0.000 0.000 TNF6 0.398 0.074 60.024 0.003 TNF7 0.011 0.005 4 0.860 0.142 TNF8 1.026 0.444 2 0.0100.001 TNF9 0.038 0.012 4 0.249 0.032 Enbrel 0.009 0.003 7 1.000 0.000Humira 0.071 0.025 8 0.103 0.059 Remicade >6.7 7

TABLE 24 % untreated RT 37° C. 50° C. 60° C. 70° C. 80° C. 90° C. TNF4100 99 99 99 98 55 34 17 TNF5 100 99 101 99 98 92 26 22 TNF6 100 103 104103 105 99 7 7 TNF7 100 100 100 98 96 66 33 40 TNF8 100 99 100 99 100 8911 8 TNF9 100 101 101 101 101 99 17 18

TABLE 25 Name SEQ ID NO Sequence TNF13(TNF1 HUM1) 76EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA VYYCARSPSGFNRGQGTQVTVSSTNF14(TNF1 HUM2) 77 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA LYYCARSPSGFNRGQGTLVTVSSTNF15(TNF2 HUM1) 78 EVQLVESGGGLVQPGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAPGKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF16(TNF2 HUM2) 79EVQLVESGGGLVQPGGSLRLSCAASGFTFSEPSGYTYTIGWFRQAPGKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF17(TNF2 HUM3) 80EVQLVESGGGLVQPGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAPGKGREFVARIYWSSGLTYYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF18(TNF2 HUM4) 81EVQLVESGGGLVQPGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAPGKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLRPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF19(TNF2 HUM5) 82EVQLVESGGGLVQPGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAPGKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTLVTVSS TNF20(TNF3 HUM1) 83EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRELLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSS TNF21(TNF3 HUM2) 84EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMGWFRQAPGKGRELLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSS TNF22(TNF3 HUM3) 85EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRELLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLKTEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSS TNF23(TNF3 HUM4) 86EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRELLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTLVTVSS ALB3(ALB1 HUM1) 87EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSSALB4(ALB1 HUM2) 88 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSSALB5(ALB1 HUM3) 89 EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSS

TABLE 26 Nanobody Induction time Yield (mg/L) TNF13 ON/28° C. 8.8 TNF14ON/28° C. 7 TNF15 Short/37° C.  7.6 TNF16 Short/37° C.  8.7 TNF17Short/37° C.  7.2 TNF18 Short/37° C.  4.8 TNF19 Short/37° C.  8 TNF20ON/28° C. 3.5 TNF21 ON/28° C. 7.5 TNF22 ON/28° C. 6 TNF23 ON/28° C. 2.8ALB3 ON/28° C. 11.8 ALB4 ON/28° C. 9 ALB5 ON/28° C. 11.7

TABLE 27 assay: L929s + Act D (5000 c/w) TNF: human TNFa @ 0.5 ng/mlEC₅₀ in nM relative potency Nanobody mean stdev # mean stdev TNF1 0.7070.265 14 0.015 0.007 TNF13 0.988 0.014 3 0.014 0.003 TNF14 0.981 0.007 30.014 0.003 TNF2 1.412 0.622 14 0.007 0.002 TNF15 1.669 1.253 4 0.0020.000 TNF16 1.898 0.192 4 0.005 0.001 TNF17 3.023 0.562 4 0.001 0.001TNF18 1.508 0.481 4 0.004 0.001 TNF19 2.191 0.941 4 0.001 0.001 TNF30.224 0.133 14 0.048 0.019 TNF20 0.380 0.080 3 0.035 0.005 TNF21 0.8890.019 3 0.015 0.003 TNF22 0.303 0.005 3 0.044 0.011 TNF23 0.3 0.011 30.044 0.011 Enbrel 0.009 0.005 45 1.002 0.011 Humira 0.079 0.043 390.097 0.069 Remicade 0.083 0.037 45 0.103 0.058

TABLE 28 % Untreated RT 37° C. 50° C. 60° C. 70° C. 80° C. 90° C. TNF13100 104 99 98 99 84 93 93 TNF14 100 98 101 95 99 96 99 90 TNF15 100 10091 99 95 90 59 46 TNF16 100 97 102 101 94 101 58 48 TNF17 100 102 98 10090 90 69 59 TNF18 100 100 101 97 91 93 63 50 TNF19 100 102 111 98 92 9160 49 TNF20 100 94 93 93 93 92 85 67 TNF21 100 98 99 101 98 96 36 40TNF22 100 102 101 105 99 93 25 31 TNF23 100 98 97 99 97 98 87 55 ALB3100 100 99 98 25 18 60 62 ALB4 100 100 100 100 99 29 61 55 ALB5 100 100100 99 94 32 61 48

TABLE 29 Name SEQ ID NO Sequence TNF1-9GS-ALB1- 90QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE 9GS-TNF1(TNF24)WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRGQGTQVTVSS TNF2-9GS-TNF2- 91QVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAP 9GS-ALB1(TNF25)GKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSEPSGYTYTIGWFRQAPGKEREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLLMNSLKPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSSTNF3-9GS-ALB1- 92 EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERE9GS-TNF3(TNF26) LLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERELLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTY WGQGTQVTVSSTNF1-30GS-TNF1- 93 QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE9GS-ALB1(TNF27) WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIG GSLSRSSQGTQVTVSSTNF3-30GS-TNF3- 94 EVQLVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERE9GS-ALB1(TNF28) LLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGSSGGGGSEVQVVESGGGLVQAGGSLSLSCSASGRSLSNYYMGWFRQAPGKERELLGNISWRGYNIYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAASILPLSDDPGWNTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS TNF30-9GS-ALB8- 417EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLE 9GS-TNF30 (TNF60)WVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSSggggsgggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSggggsgggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSS TNF33-9GS-ALB8- 418EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRE 9GS-TNF33 (TNF62)LLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLRPEDTAVYYCAASILPLSDDPGWNTYWGQGTLVTVSSggggsgggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSggggsgggsEVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRELLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLRPEDTAVYYCAASILPLSDDPGWNTY WGQGTLVTVSS

TABLE 30 ID Format TNF24 TNF1-9GS-ALB1-9GS-TNF1 TNF25TNF2-9GS-TNF2-9GS-ALB1 TNF26 TNF3-9GS-ALB1-9GS-TNF3 TNF27TNF1-30GS-TNF1-9GS-ALB1 TNF28 TNF3-30GS-TNF3-9GS-ALB1

TABLE 31 Nanobody Induction time Yield (mg/L) TNF24  ON/28° C. 1.7 TNF25short/37° C. 0.445 TNF26 short/37° C. 0.167 TNF27  ON/28° C. 2.2 TNF28short/37° C. 1

TABLE 32 assay: L929s + Act D (5000 c/w) TNF: human TNFa @ 0.5 ng/mlEC₅₀ in nM relative potency Nanobody mean stdev # mean stdev TNF24 0.0110.003 11 0.878 0.248 TNF25 0.018 0.008 14 0.603 0.243 TNF26 0.020 0.00914 0.583 0.210 TNF27 0.012 0.003 3 0.810 0.037 TNF28 0.021 0.008 6 0.5480.360 Enbrel 0.009 0.005 45 1.002 0.011 Humira 0.079 0.043 39 0.0970.069 Remicade 0.083 0.037 45 0.103 0.058

TABLE 33 Human albumin KD (nM) ka (1/Ms) kd (1/s) 6A6 (ALB1) 0.571.11E+6  6.30E−4  1C2-GS-6A6-GS-1C2 (TNF24) 11 2.26E+05 2.48E−031G5-GS-1G5-GS-6A6 (TNF25) 7.2 2.91E+05 2.10E−03 5F10-GS-6A6-GS-5F10(TNF26) 7.3 2.81E+05 2.05E−03 1C2-GS6-1C2-GS-6A6 (TNF27) 8.9 3.19E+052.84E−03 5F10-GS6-5F10-GS-6A6 (TNF28) 14 1.55E+05 2.13E−03

TABLE 34 % untreated RT 37° C. 50° C. 60° C. 70° C. 80° C. 90° C. TNF24100 100 99 98 5 3 8 18 TNF25 100 nd 103 102 95 5 4 6 TNF26 100 109 115112 107 10 8 10 TNF27 100 102 103 102 22 9 26 34 TNF28 100  97 99 99 663 6 10

TABLE 35 Name SEQ ID NO Sequence TNF29(TNF1 HUM1) 95EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTA VYYCARSPSGFNRGQGTLVTVSSTNF30(TNF1 HUM2) 96 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTA VYYCARSPSGFNRGQGTLVTVSSTNF31(TNF2 HUM1) 97 EVQLVESGGGLVQPGGSLRLSCAASGFTFSEPSGYTYTIGWFRQAPGKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLRPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTQVTVSS TNF32(TNF2 HUM2) 98EVQLVESGGGLVQPGGSLRLSCAASGFTFSEPSGYTYTIGWFRQAPGKGREFVARIYWSSGLTYYADSVKGRFTISRDIAKNTVDLQMNSLRPEDTAVYYCAARDGIPTSRSVGSYNYWGQGTLVTVSS TNF33(TNF3 HUM1) 99EVQLVESGGGLVQPGGSLRLSCAASGRSLSNYYMGWFRQAPGKGRELLGNISWRGYNIYYKDSVKGRFTISRDDSKNTIYLQMNSLRPEDTAVYYCAASILPLSDDPGWNTYWGQGTLVTVSS ALB6(ALB1 HUM1) 100EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTLVTVSSALB7(ALB1 HUM2) 101 EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSSALB8(ALB1 HUM3) 102 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSSALB9(ALB1 HUM4) 103 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSSALB10(ALB1 HUM5) 104 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTA VYYCTIGGSLSRSGQGTLVTVSS

TABLE 36 Nanobody ™ Induction time yield TNF29 ON/28° C. 2.1 mg/L TNF30ON/28° C. 2.7 mg/L TNF31 ON/28° C.  2 mg/L TNF32 ON/28° C. 1.5 mg/LTNF33 ON/28° C. 0.5 mg/L

TABLE 37 assay: L929s + Act D (5000 c/w) TNF: human TNFa @ 0.5 ng/mlEC₅₀ in nM relative potency V_(HH) mean stdev # mean stdev TNF1 0.7070.265 14 0.015 0.007 TNF13 0.988 0.014 3 0.014 0.003 TNF14 0.981 0.007 30.014 0.003 TNF29 1.336 1 0.013 TNF30 0.985 1 0.017 TNF2 1.412 0.622 140.007 0.002 TNF15 5.896 1.253 4 0.002 0.000 TNF16 2.422 0.192 4 0.0050.001 TNF17 7.555 0.562 4 0.001 0.001 TNF18 3.134 0.481 4 0.004 0.001TNF19 7.372 0.941 4 0.001 0.001 TNF31 2.195 1 0.008 TNF32 2.506 1 0.007TNF3 0.224 0.133 14 0.048 0.019 TNF20 0.380 0.080 3 0.035 0.005 TNF210.889 0.019 3 0.015 0.003 TNF22 0.303 0.005 3 0.004 0.011 TNF23 0.30.011 3 0.04 0.011 TNF33 0.3 1 0.057 Enbrel 0.009 0.005 45 1.002 0.011Humira 0.079 0.043 39 0.097 0.069 Remicade 0.083 0.037 45 0.103 0.058

TABLE 38 untreated RT 37° C. 50° C. 60° C. 70° C. 80° C. 90° C. TNF29100 100 100 100 100 96 91 89 TNF30 100 100 100 99 100 96 92 89 TNF31 100100 100 98 91 84 56 43 TNF32 100 99 98 97 87 78 45 39 TNF33 100 98 97 9794 91 79 49

TABLE 39 assay: alphaKYM (10000 c/w) TNF: human TNFa @ 1 ng/ml NanobodyEC₅₀ in nM TNF1 2.466 TNF2 4.236 TNF3 0.655 TNF4 0.069 TNF5 0.008 TNF60.121 TNF7 0.009 TNF8 0.013 TNF9 0.020 Enbrel 0.040 Humira 0.103Remicade 0.100 Results from WO 04/41862 Nanobody SEQ ID No EC₅₀ in nM 1A1 100 3E 4 12 3G 5 20 Remicade 0.080

TABLE 40 M13_rev SEQ ID GGATAACAATTTCACACAGG NO: 421 Rev_9GlySer_L108SEQ ID TCAGTAACCTGGATCCGCCACCGCT NO: 422 GCCTCCACCGCCTGAGGAGACGGTG ACCAGFor_GS/Short SEQ ID AGGTTACTGAGGATCCGAGGTGCAGC NO: 423 TGGTGGAGTCTGGRev_15BspEI_L108 SEQ ID TCAGTAACCTTCCGGAACCGCCACC NO: 424GCCTGAGGAGACGGTGACAAG For_BspEI SEQ ID AGGTTACTGATCCGGAGGCGGTAGC NO: 425GAGGTGCAGCTGGTGGAGTCTGG M13_for SEQ ID CACGACGTTGTAAAACGAC NO: 426

TABLE 41 Sequence Reverse primer PiRevhumNot/ SEQ IDATGGTGGTGTGCGGCCGCCTATTATGA a40c (Not1) NO: 427 GGAGACGGTGACCAGGForward primer Pi2for (Xho1) SEQ ID AGGGGTATCTCTCGAGAAAAGAGAGGT NO: 428GCAGCTGGTGGAGTCTGG

TABLE 42 Human TNFα EC₅₀ in nM VHH mean stdev Number of assays TNF600.010 0.002 6 Enbrel 0.014 0.009 33 Humira 0.141 0.074 33 Remicade 0.1200.037 33

TABLE 43 human albumin rhesus albumin TNF60 K_(D) (nM) 24.4 24.1 k_(on)(1/Ms) 2.05E+05 2.09E+05 k_(off) (1/s) 5.02E−03 5.04E−03 TNF24 K_(D)(nM) 11   Nd k_(on) (1/Ms) 2.26E+05 Nd k_(off) (1/s) 2.48E−03 Nd

TABLE 44 PiForLong SEQ ID GCTAAAGAAGAAGGGGTATCTCTC NO: 429GAGAAAAGAGAGGTGCAGCTGGTG GAGTCTGG Rev_30GlySer_L108 SEQ IDTCAGTAACCTGGATCCCCCGCCA NO: 430 CCGCTGCCTCCACCGCCGCTACCCCCGCCACCGCTGCCTCCACCGCCT GAGGAGACGGTGACAAG For_GlySer SEQ IDAGGTTACTGAGGATCCGGCGGTG NO: 431 GAGGCAGCGGTGGCGGGGGTAGCGAGGTGCAGCTGGTGGAGTCTGG PiRevCys1hum SEQ ID ATGGTGGTGTGAATTCTTATTAGCNO: 432 AGGAGACGGTGACAAGG PiRevCys2hum SEQ ID ATGGTGGTGTGAATTCTTATTAGCNO: 433 AACCTCCACCTGAGGAGACG GTGACAAGG AOXIFor SEQ IDGACTGGTTCCAATTGACAAGC NO: 434 AOXIRev SEQ ID GCAAATGGCATTCTGACATCCNO: 435

TABLE 45 Human TNFα EC₅₀ in nM VHH mean stdev Number of assays TNF10.748 0.153 27 TNF55-PEG40 0.004 0.001 8 TNF55-PEG60 0.004 0.002 6TNF55-Biotine 0.012 0.003 5 TNF56-PEG40 0.006 0.003 57 TNF56-PEG60 0.0050.003 7 TNF56-Biotine 0.017 0.009 13 Enbrel 0.013 0.006 71 Humira 0.1270.058 67 Remicade 0.144 0.061 67

TABLE 46 DS534 P4 DS592 P4 DS605 P3 KM05-179 P4 p[IC]50 Etanercept 9.559.49 9.51 9.51 Accipiter 9.88 9.44 9.37 9.27 pM Etanercept 282 324 309309 Accipiter 132 363 427 537

TABLE 47 Total WBC count (×10⁶/mL) Dose Group ALX0071 Etanercept CMCvehicle 0.86 ± 0.09 0.1 μg human TNFα 3.72 ± 0.21 0.0625 mg/kg 3.03 ±0.6  — 0.125 mg/kg  1.23 ± 0.3** 2.40 ± 0.39 0.25 mg/kg  1.37 ± 0.17*2.47 ± 0.54 0.5 mg/kg — 2.19 ± 0.10 *P < 0.05, **P < 0.01 vs 0.1 μghuman TNFα

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

The invention claimed is:
 1. A polypeptide comprising the amino acidsequence of: DVQLVESGGGLVQPGGSLKLSCAASGFDFSX³¹X³²WMYWVRQAPGKELEWLSEINTNGLITX⁵⁹YX⁶¹DSVKGRFTVSRNNAANKMYLELTRLEPEDTALYYCARX⁹⁹X¹⁰⁰X¹⁰¹GX¹⁰³NKGQGTQVTVSS (SEQ ID NO: 478), wherein: the amino acid of X³¹ is a polaramino acid selected from the group consisting of G, T, C, N, Q, Y, K, R,H, D and E; the amino acid of X³² is a polar amino acid selected fromthe group consisting of G, S, T, C, N, Q, Y, K, R, H, D and E; the aminoacid of X⁵⁹ is a polar, positively charged amino acid selected from thegroup consisting of K, R, and H; the amino acid of X⁶¹ is a smallaliphatic, nonpolar or slightly polar residue selected from the groupconsisting of A, S, T, P, and G; the amino acid of X⁹⁹ is a polar,uncharged amino acid selected from the group consisting of G, S, T, C,N, Q, and Y; the amino acid of X¹⁰⁰ is any amino acid; the amino acid ofX¹⁰¹ is a polar amino acid selected from the group consisting of G, S,T, C, N, Q, Y, K, R, H, D, and E; and the amino acid of X¹⁰³ is anonpolar, uncharged amino acid selected from the group consisting of A,V, L, I, F, M, W, and P.
 2. The polypeptide of claim 1, wherein thepolypeptide binds human TNFα.
 3. A pharmaceutical composition comprisingthe polypeptide of
 1. 4. The pharmaceutical composition of claim 3,further comprising one or more excipients.
 5. A method for treating aTumor Necrosis Factor-α (TNF-α) related disorder comprisingadministering, to a subject in need thereof, an effective amount of thepharmaceutical composition of claim
 3. 6. The method of claim 5, whereinthe disorder is Crohn's disease, ulcerative colitis or inflammatorybowel syndrome.